Chemo-immunotherapy method

ABSTRACT

A method of treating or preventing disease, said method comprising administering to a subject, simultaneously, sequentially or separately, an antigen and a chemotherapeutic agent or agents comprising the steps of: administering the chemotherapeutic agent or agents, and administering an antigen up to 6 weeks after the chemotherapeutic agent.

FIELD OF THE INVENTION

The present invention relates to a combination therapy for treating anindividual with an antigen to elicit an antigen-specific immune responsealong with treating that individual with a chemotherapeutic agent.

BACKGROUND TO THE INVENTION

Treatment of patients with advanced cancers is generally bychemotherapy. However, for solid tumours, in particular, it is rarelycurative and additional routes of therapy are required.

Recently progress in human immunobiotechnology has opened up the fieldof immunotherapy as a new approach to cancer treatment. Specificimmunization against a target antigen has been achieved in some patientswith a number of different anticancer vaccines, but improved long termresponses are desirable.

Accordingly, there remains a need for improved therapy regimes.

As chemotherapy has a number of observed detrimental effects on theimmune system, chemotherapy and immunotherapy are regarded as unrelatedor, more commonly, antagonistic forms of therapy. This is because, mostchemotherapies kill target cells by apoptosis and this mode of celldeath has been regarded immunologically as either non-stimulatory orable to produce immune tolerance—a state where T cells can no longerrespond to the presented antigen by mounting an immune resp

a common side effect of chemotherapies is the induction of lyn

reduction in lymphocytes and this is assumed to be detrimental

immune response.

SUMMARY OF THE INVENTION

The present invention relates to a combination treatment comprisingadministering an antigen, to elicit an immune response specific to thatantigen, as well as a chemotherapeutic agent or agents. In particular,the present invention is based on the surprising finding that treatmentto stimulate an immune response against an antigen by administering anantigen is enhanced by treatment with a chemotherapeutic agent oragents.

Accordingly, in a first aspect of the invention, there is provided apharmaceutical composition comprising an antigen and a chemotherapeuticagent or agents and a pharmaceutically acceptable carrier, diluent orexcipient.

Suitably, such a composition is provided as the two components forseparate, simultaneous or subsequent administration in a form such as akit.

Suitably, the antigen is an antigen to which it would be desirable toelicit an immune response. Such antigens include, for example,inactivated, attenuated or nonpathogenic strains of pathogens which areused as antigens to induce immunity against diseases caused by pathogenssuch as typhoid, polio, measles, mumps, rubella and tuberculosis,allergenic proteins from pollen and other allergenic material which areisolated and used to immunise a patient, antigenic components ofpathogenic organisms such as Haemophilus influenza B, Hepatitis B and soforth.

In another embodiment, the antigen may be one which can be used in amethod for contraceptive such as a method for inducing an immuneresponse which is generated a sperm or egg specific protein. Forexample, the antigen may be a zona pellucida protein.

In one embodiment, the antigen is a tumor associated antigen. A suitabletumour associated antigen (TAAs) includes 5T4. Other suitable antigensinclude TAAs in the following classes: cancer testis antigens (egHOM-MEL-40), differentiation antigens (eg HOM-MEL-55), overexpressedgene products (HOM-MD-21), mutated gene products (NY-COL-2), splicevariants (HOM-MD-397), gene amplification products (HOM-NSCLC-11) andcancer related autoantigens (HOM-MEL-2.4) as reviewed in Cancer Vaccinesand Immunotherapy (2000) Eds Stern, Beverley and Carroll, CambridgeUniversity Press, Cambridge. Further examples include, MART-1 (MelanomaAntigen Recognised by T cells-1) MAGE-A (MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A10, MAGE-A12), MAGE B(MAGE-B1-MAGE-B24), MAGE-C (MAGE-C1/CT7, CT10), GAGE (GAGE-1, GAGE-8,PAGE-1, PAGE-4, XAGE-1, XAGE-3), LAGE (LAGE-1a(1S), -1b(1L), NY-ESO-1),SSX (SSX1-SSX-5), BAGE, SCP-1, PRAME (MAPE), SART-1, SART-3, CTp11,TSP50, CT9/BRDT, gp100, MART-1, TRP-1, TRP-2, MELAN-A/MART-1,Carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), MUCIN(MUC-1) and Tyrosinase. TAAs are reviewed in Cancer Immunology (2001)Kluwer Academic Publishers, The Netherlands. Additional tumourassociated antigens include Her 2, survivin and TERT.

By “antigen” is included a means for providing an antigen protein orpeptide to be introduced into an individual. As described herein, anantigen may be provided through delivering a peptide or protein orthrough delivering a nucleic acid encoding a peptide or protein.

By “antigen” in the context of the present invention it is also meant toincorporate an antigenic peptide derived from an antigen. In particular,“tumour associated antigen” is intended to encompass a peptide derivedfrom a tumour associated antigen.

An antigen such as a tumour associated antigen can be provided for useas a medicament in a number of different ways. It can be administered aspart of a viral vector. A number of suitable viral vectors will befamiliar to those skilled in the art and include a number of vectorsdescribed herein.

The TroVax® vaccine (TroVax is a registered US, and European Communitytrade mark of Oxford Biomedica plc) comprises a viral vector derivedfrom MVA which has been modified to express the tumour associatedantigen, 5T4. In one embodiment the tumour antigen for use in thecombination of the invention is 5T4 and is provided by the TroVax®vaccine.

Suitable chemotherapeutic agents include any conventional agents. In oneembodiment, the chemotherapeutic agent is selected from irinotecan,fluorouracil, leucovorin, and oxaliplatin. In another embodiment a“chemotherapeutic agent” can include a combination of chemotherapeuticagents in a therapy such as FOLFOX or IFL.

In another aspect there is provided a use of a composition according tothe present invention in the manufacture of a medicament for thetreatment of a disease. Suitably, where the antigen is a tumourassociated antigen, the disease is cancer.

In a further aspect there is provided a pharmaceutical productcomprising an antigen and a chemotherapeutic agent or agents forsimultaneous, sequential or separate use in therapy. Suitably theantigen is a tumour associated antigen.

By “simultaneous” is meant that two agents are administeredconcurrently. By “sequential” is meant that the two agents areadministered one after the other within a time frame such that they areboth available to act therapeutically within the same time frame. Theoptimum time interval between administering the two agents will varydepending on the precise nature of the method for delivering the tumourantigen.

The term “separate” is used to mean that the gap between theadministration of the first agent and that of the second is substantial.

In one embodiment, chemotherapy is administered before administration ofthe antigen. Suitably, chemotherapy is administered 10 weeks beforeadministration of the antigen, preferably less than 10 weeks and, morepreferably in about 2 weeks before administration of antigen. In apreferred embodiment, chemotherapy is administered 2 weeks beforeadministration of the antigen.

In one embodiment, the antigen is administered in advance of thebeginning of chemotherapy and then again following chemotherapy. Inanother embodiment, the antigen is administered during chemotherapy aswell as before and/or following chemotherapy.

In one embodiment, the antigen is administered at least 24 hours,preferably 48 hours after chemotherapy. Preferably, administration ofthe antigen takes place up to 8 weeks, preferably up to 6 weeks, evenmore preferred between 4 and 6 weeks after chemotherapy.

In one embodiment the antigen is administered when the subject to betreated has a reduced CD4⁺CD25⁺ Treg cell count or reduced CD4⁺CD25⁺Treg cell function.

By “reduced CD4⁺CD25⁺ Treg cell count” is meant a CD4⁺CD25⁺ Treg cellcount which is lower then the CD4⁺CD25⁺ Treg cell count determined priorto administering a CD4⁺CD25⁺ Treg cell count lowering agent, such as achemotherapeutic agent or agents, or Ontak. Preferably the CD4⁺CD25⁺Treg cell level is at least 15% 30%, 50%, 70%, 90% lower then theCD4⁺CD25⁺ Treg cell level determined prior to administering theCD4⁺CD25⁺ Treg cell count lowering agent (for example a chemotherapeuticagent or agents or Ontak), Most preferably, the vaccination willcoincide with the period when the chemotherapy has caused a maximumdepletion of CD4⁺CD25⁺ Treg cell.

By “reduced CD4⁺CD25⁺ Treg cell function” is meant a CD4⁺CD25⁺ Treg cellfunction which is lower then the CD4⁺CD25⁺ Treg cell function determinedprior to administering a CD4⁺CD25⁺ Treg cell function lowering agent,such as a chemotherapeutic agent, or Ontak. Preferably the CD4⁺CD25⁺Treg cell function is at least 15% 30%, 50%, 70%, 90% lower then theCD4⁺CD25⁺ Treg cell function determined prior to administering theCD4⁺CD25⁺ Treg cell function lowering agent (for example achemotherapeutic agent or agents or Ontak), Most preferably, thevaccination will coincide with the period when the chemotherapy hascaused a maximum reduction of CD4⁺CD25⁺ Treg cell function.

In one embodiment, there is provided a pharmaceutical product inaccordance with the invention in the form of a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier, diluent orexcipient.

In another aspect there is provided a method of treating or preventing adisease, said method comprising administering to a subjectsimultaneously, sequentially or separately, an antigen and achemotherapeutic agent.

Suitably, the method of treating an individual comprises the steps of:

a) administering chemotherapyb) administering an antigen.

In one embodiment, the method further comprises administering an antigenprior to administering chemotherapy. In another embodiment, the methodfurther comprises adminstering an antigen during the same time frame asadministering chemotherapy.

In another aspect there is provided a method of treating cancer, saidmethod comprising administering to a subject simultaneously,sequentially or separately, a tumour associated antigen and achemotherapeutic agent.

Suitably, the method of treating an individual comprises the steps of

a) administering a tumour antigen to elicit an antitumour response,b) administering chemotherapyc) administering a tumour antigen.

In one embodiment the method further comprises the step of determiningthe CD4⁺CD25⁺ Treg cell count or CD4⁺CD25⁺ Treg cell function. Inanother embodiment the method comprises determining the CD4⁺CD25⁺ Tregcell count or reduced CD4⁺CD25⁺ Treg cell function prior to and/orduring and/or after chemotherapy.

In a further aspect there is provided a use of an antigen in thepreparation of a medicament for use in the treatment or prevention of adisease wherein said treatment comprises administering to a subjectsimultaneously, sequentially or separately a chemotherapeutic agent oragents and an antigen.

In another aspect there is provided a use of an antigen and achemotherapeutic agent or agents in the preparation of a medicament foruse in the treatment or prevention of a disease.

In another aspect there is provided a use of an antigen in thepreparation of a medicament for use in the treatment or prevention of adisease wherein said treatment is for use in combination therapy with achemotherapeutic agent.

In another aspect there is provided a use of a chemotherapeutic agent oragents in the preparation of a medicament for use in the treatment orprevention of a disease wherein said treatment is for use in combinationtherapy with an antigen.

In a further aspect there is provided a use of an antigen and achemotherapeutic agent or agents in the manufacture of a medicament forsimultaneous, separate or sequential use in the treatment or preventionof a disease wherein the administration pattern comprises administeringthe antigen prior to administering chemotherapy and administering theantigen after chemotherapy.

In another aspect there is provided a use of an antigen in thepreparation of a medicament for administering to a mammal so as toinduce an immune response to an antigen wherein the administration ofthe antigen comprises administering the antigen prior to and/or duringand/or after administration of a chemotherpeutic agent in the treatmentor prevention of a disease.

Suitably, in one embodiment of any of the above aspects, the antigen isa tumour associated antigen and the disease is cancer.

In a further aspect of the invention, there is provided a method ofenhancing an immune response to an antigen by treating with chemotherapybefore and/or during and/or after sensitising to the antigen.

In another aspect there is provided a kit comprising a means foradministering an antigen in combination with a chemotherapeutic agent oragents for administration.

Other aspects of the present invention are presented in the accompanyingclaims and in the following description and discussion. These aspectsare presented under separate section headings. However, it is to beunderstood that the teachings under each section heading are notnecessarily limited to that particular section heading.

DETAILED DESCRIPTION OF THE INVENTION Antigens Tumour AssociatedAntigens (TAAS)

TAAs have been characterised either as membrane proteins or alteredcarbohydrate molecules of glycoproteins and glycolipids, however theirfunctions remain largely unknown. One TAA family, the transmembrane 4superfamily (TM4SF), usually has four well-conserved membrane-spanningregions, certain cysteine residues and short sequence motifs. There isevidence that TM4SF antigens exist in close association with otherimportant membrane receptors including CD4 and CD8 of T cells (Imai &Yoshie (1993) J. Immunol. 151, 6470-6481). It has also been suggestedthat TM4SF antigens may play a role in signal transduction which inturn, affects cell development, activation and motility. Examples ofTM4SF antigens include human melanoma-associated antigen ME491, humanand mouse leukocyte surface antigen CD37, and human lymphoblasticleukemia-associated TALLA-1 (Hotta, H. et al. (1988) Cancer Res. 48,2955-2962; Classon, B. J. et al. (1989) J. Exp. Med. 169: 1497-1502;Tomlinson, M. G. et al. (1996) Mol. Immun. 33: 867-872; Takagi, S. etal. (1995) Int. J. Cancer 61: 706-715).

Further examples of TAAs also include, but are not limited to, TAAs inthe following classes: cancer testis antigens (HOM-MEL-40),differentiation antigens (HOM-MEL-55), overexpressed gene products(HOM-MD-21), mutated gene products (NY-COL-2), splice variants(HOM-MD-397), gene amplification products (HOM-NSCLC-11) and cancerrelated autoantigens (HOM-MEL-2.4) as reviewed in Cancer Vaccines andImmunotherapy (2000) Eds Stern, Beverley and Carroll, CambridgeUniversity Press, Cambridge. Further examples include, MART-1 (MelanomaAntigen Recognised by T cells-1) MAGE-A (MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A10, MAGE-A12), MAGE B(MAGE-B1-MAGE-B24), MAGE-C (MAGE-C1/CT7, CT10), GAGE (GAGE-1, GAGE-8,PAGE-1, PAGE-4, XAGE-1, XAGE-3), LAGE (LAGE-1a(1S), -1b(1L), NY-ESO-1),SSX (SSX1-SSX-5), BAGS, SCP-1, PRAME (MAPE), SART-1, SART-3, CTp11,TSP50, CT9/BRDT, gp100, MART-1, TRP-1, TRP-2, MELAN-A/MART-1,Carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), MUCIN(MUC-1) and Tyrosinase. TAAs are reviewed in Cancer Immunology (2001)Kluwer Academic Publishers, The Netherlands.

In one embodiment, the tumour associated antigen is 5T4.

5T4

5T4 has been previously characterised, for example, in WO89/07947. Thesequence of human 5T4 appears in GenBank at accession no. Z29083. The5T4 antigen may come from a different species, such as murine 5T4(WO00/29428), canine 5T4 (WO01/36486) or feline 5T4. The antigen mayalso be derived from a naturally occurring variant of 5T4 found within aparticular species, preferably a mammal. Such a variant may be encodedby a related gene of the same gene family, by an allelic variant of aparticular gene, or represent an alternative splicing variant of the 5T4gene. 5T4 and its use has also been described in EP1036091.

A peptide epitope derived from 5T4 from a different species or a splicevariant may have a different amino acid sequence from the analogoushuman wild-type 5T4 peptide epitope. However, as long as the peptideretains the same qualitative binding specificity as the human peptide(i.e. it binds in the peptide binding groove of an MHC molecule of thesame haplotype) then it is still an epitope in accordance with thepresent invention.

Immunogenic Peptides Derived from Antigens

“Antigens” include peptide epitopes derived from specific antigenicproteins including tumour associated antigens. Suitable epitopes includeT cell epitopes. Suitably said peptides are “immunogenic peptides” i.e.they are capable of stimulating an anti-tumour associated antigen immuneresponse. Such an immune response includes a cytotoxic T cell responsefor peptides as well as a cytotoxic T cell response and/or antibodyresponse for protein antigens in general.

In this respect, the term “peptide” is used in the normal sense to meana series of residues, typically L-amino acids, connected one to theother typically by peptide bonds between the α-amino and carboxyl groupsof adjacent amino acids. The term includes modified peptides andsynthetic peptide analogues.

A T cell epitope is a short peptide derivable from a protein antigen.Antigen presenting cells can internalise antigen and process it intoshort fragments which are capable of binding MHC molecules. Thespecificity of peptide binding to the MHC depends on specificinteractions between the peptide and the peptide-binding groove of theparticular MHC molecule.

Peptides which bind to MHC class I molecules (and are recognised by CD8+T cells) are usually between 6 and 12, more usually between 8 and 12amino or 8 and 10 amino acids in length. Typically, peptides are 9 aminoacids in length. The amino-terminal amine group of the peptide makescontact with an invariant site at one end of the peptide groove, and thecarboxylate group at the carboxy terminus binds to an invariant site atthe other end of the groove. Thus, typically, such peptides have ahydrophobic or basic carboxy terminus and an absence of proline in theextreme amino terminus. The peptide lies in an extended conformationalong the groove with further contacts between main-chain atoms andconserved amino acid side chains that line the groove. Variations inpeptide length are accommodated by a kinking in the peptide backbone,often at proline or glycine residues.

Peptides which bind to MHC class II molecules are usually at least 10amino acids, for example about 13-18 amino acids in length, and can bemuch longer. These peptides lie in an extended conformation along theMHC II peptide-binding groove which is open at both ends. The peptide isheld in place mainly by main-chain atom contacts with conserved residuesthat line the peptide-binding groove.

Antigenic peptides of the present invention may be made using chemicalmethods (Peptide Chemistry, A practical Textbook. Mikos Bodansky,Springer-Verlag, Berlin). For example, peptides can be synthesized bysolid phase techniques (Roberge J Y et al (1995) Science 269: 202-204),cleaved from the resin, and purified by preparative high performanceliquid chromatography (e.g., Creighton (1983) Proteins Structures AndMolecular Principles, WH Freeman and Co, New York N.Y.). Automatedsynthesis may be achieved, for example, using the ABI 43 1 A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer.

The peptide may alternatively be made by recombinant means, or bycleavage from a longer polypeptide. For example, the peptide may beobtained by cleavage from a full-length protein such as full length 5T4.The composition of a peptide may be confirmed by amino acid analysis orsequencing (e.g., the Edman degradation procedure).

The term “peptide epitope” encompasses modified peptides. For exampletumour associated antigen peptides may be mutated, by amino acidinsertion, deletion or substitution, so long as the MHCbinding-specificity of the wild-type tumour associated antigen peptideis retained. In a preferred embodiment the modified epitope has greateraffinity for the peptide binding groove. Preferably the peptide contains5 or fewer mutations from the wild-type sequence, more preferably 3 orfewer, most preferably 1 or 0 mutations.

Alternatively (or in addition) modifications may be made withoutchanging the amino acid sequence of the peptide. For example, D-aminoacids or other unnatural amino acids can be included, the normal amidebond can be replaced by ester or alkyl backbone bonds, N- or C-alkylsubstituents, side chain modifications, and constraints such asdisulphide bridges and side chain amide or ester linkages can beincluded. Such changes may result in greater in vivo stability of thepeptide, and a longer biological lifetime.

Other forms of modification included posttranslational modificationssuch as phosphorylation and glycosylation of the peptides.Posttranslational modified peptides induce essentially the same immuneresponse as other peptides according to the invention. Certainmodifications might lead to an increase in the induced immune responsewhereas others would decrease the response. Other modifications includethe addition or removal of a glycosylation or phosphohorylations site tochange or modulate the immune response stimulated by these peptides.

Modification of epitopes may be performed based on predictions for moreefficient T-cell induction derived using the program “Peptide BindingPredictions” devised by K. Parker (NIH) which may be found athttp://www-bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform (seealso Parker, K. C et al. 1994. J. Immunol. 152:163).

A “modified” antigenic peptide epitope includes peptides which have beenbound or otherwise associated to transporter peptides or adjuvants, inorder to increase their ability to elicit an immune response. Forexample, peptides may be fused to TAP independent transporter peptidesfor efficient transport to HLA and interaction with HLA molecules toenhance CTL epitopes (for review see Yewdell et al., 1998 J Immunother21:127-31; Fu et al., (1998) J Virol 72:1469-81).

In a further embodiment, antigens or peptides derived from such antigensmay be fused to hepatitis B core antigen to enhance T helper andantibody responses (Schodel et al., 1996 Intervirology 39:104-10).

To be an epitope, the peptide should be capable of binding to thepeptide-binding groove of a MHC class I or II molecule and be recognisedby a T cell.

Cell surface presentation of peptides derived from a given antigen isnot random and tends to be dominated by a small number of frequentlyoccurring epitopes. The dominance of a particular peptide will depend onmany factors, such as relative affinity for binding the MHC molecule,spatio-temporal point of generation within the APC and resistance todegradation. The epitope hierarchy for an antigen is thought to changewith progression of an immune response. After a primary immune responseto the immunodominant peptides, epitope “spreading” may occur tosub-dominant determinants (Lehmann et al (1992) Nature 358:155-157).

For any given antigen, cryptic epitopes may also exist. Cryptic epitopesare those which can stimulate a T cell response when administered as apeptide but which fail to produce such a response when administered as awhole antigen. It may be that during processing of the antigen intopeptides in the APC the cryptic epitope is destroyed.

The peptide for use in the invention may be an immunodominant epitope, asub-dominant epitope or a cryptic epitope.

Epitopes for an antigen may be identified by measuring the T cellresponse to overlapping peptides spanning a portion of the antigen whenpresented by APC. Such studies usually result in “nested sets” ofpeptides, and the minimal epitope for a particular T cell line/clone canbe assessed by measuring the response to truncated peptides.

The minimal epitope for an antigen may not be the best epitope forpractical purposes. It may well be that amino acids flanking the minimalepitope will be required for optimal binding to the MHC.

The peptides are tested in an antigen presentation system whichcomprises antigen presenting cells and T cells. For example, the antigenpresentation system may be a murine splenocyte preparation, apreparation of human cells from tonsil or PBMC. Alternatively, theantigen presentation system may comprise a particular T cell line/cloneand/or a particular antigen presenting cell type.

T cell activation may be measured via T cell proliferation (for exampleusing ³H-thymidine incorporation) or cytokine production. Activation ofTH1-type CD4+ T cells can, for example be detected via IFNγ productionwhich may be detected by standard techniques, such as an ELISPOT assay.

Polyepitope String

It has been found that a particularly effective way to induce an immuneresponse to an antigen is by the use of a polyepitope string, whichcontains a plurality of antigenic epitopes from one or more antigenslinked together. For example, for malaria, a polyepitope string ofmainly malaria (P. falciparum) CD8 T cell peptide epitopes has beendescribed which also expresses CD4 T cell epitopes from tetanus toxoidand from the 38 Kd mycobacterial antigen of various strains of M.tuberculosis and M. bovis.

Accordingly, the tumour associated antigen for use in the presentinvention may be a polyepitope string comprising at least one peptidefrom a tumour associated antigen. Suitably a polyepitope string is madeup of at least one, two, three, four or more peptide epitopes. Thestring may also comprise another epitope derivable from an antigen suchas the 5T4 antigen or an epitope from another antigen—such as anotherTAA—or combinations thereof. A polyepitope string may optionallycomprise additional intervening amino acids between the different tumourantigen epitopes. Suitably epitopes are joined by additional sequencesthat are absent from the full length protein.

Cell Penetrators

The present invention also provides the use of an antigen or a peptideepitope thereof, or a polyepitope string in association with a cellpenetrator.

Antigen presenting cells (such as dendritic cells) pulsed with peptideshave proven effective in enhancing antitumour immunity (Celluzzi et al(1996) J. Exp. Med. 183 283-287; Young et al (1996) J. Exp. Med. 1837-11). It has been shown that it is possible to prolong the presentationof a peptide by dendritic cells (and thus enhance antitumour immunity)by linking it to a cell penetrating peptide (CPP) (Wang and Wang (2002)Nature Biotechnology 20 149-154).

A cell penetrator may be any entity which enhances the intracellulardelivery of the peptide/polyepitope string to the antigen presentingcell. For example, the cell penetrator may be a lipid which, whenassociated with the peptide, enhances its capacity to cross the plasmamembrane. Alternatively, the cell penetrator may be a peptide. Severalcell penetrating peptide (CPPs) have been identified from proteins,including the Tat protein of HIV (Frankel and Pabo (1988) Cell 551189-1193), the VP22 protein of HSV (Elliott and O'Hare (1997) Cell 88223-233) and fibroblast growth factor (Lin et al (1995) J. Biol. Chem.270 14255-14258).

The term “associated with” is intended to include direct linkage, forexample by a covalent bond. Examples of covalent bonds for linking aminoacids include disulphide bridges and peptide bonds. In a preferredembodiment, the peptide/polyepitope string and a CPP are linked by apeptide bond to create a fusion protein.

The term also includes non-covalent linkage, such as association byelectrostatic bonding, hydrogen bonding and van der Waals forces. Thecell penetrator and peptide/polyepitope string may be associated withoutcovalent or non-covalent bonding. For example the cell penetrator may bea lipid which encapsulates the peptide/polyepitope string (e.g. a.liposome).

Compositions for Administering Tumour Associated Antigens Vector System

A nucleic acid sequence encoding an antigen for use in the presentinvention may be delivered or administered to a mammal such as a humanpatient by way of a vector system.

As used herein, a “vector” may be any agent capable of delivering ormaintaining nucleic acid in a host cell, and includes viral vectors,plasmids, naked nucleic acids, nucleic acids complexed with polypeptideor other molecules and nucleic acids immobilised onto solid phaseparticles. Such vectors are described in detail below. It will beunderstood that the present invention, in its broadest form, is notlimited to any specific vector for delivery of the tumour associatedantigen-encoding nucleic acid.

Nucleic acids encoding antigens, epitopes and polyepitope strings inaccordance with the present invention can be delivered or administeredby viral or non-viral techniques.

Non-viral delivery systems include but are not limited to DNAtransfection methods. Here, transfection includes a process using anon-viral vector to deliver a gene encoding an antigen to a targetmammalian cell.

Typical transfection methods include electroporation, nucleic acidbiolistics, lipid-mediated transfection, compacted nucleic acid-mediatedtransfection, liposomes, immunoliposomes, lipofectin, cationicagent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology1996 14; 556), multivalent cations such as spermine, cationic lipids orpolylysine, 1,2,-bis(oleoyloxy)-3-(trimethylammonio) propane(DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 NatureBiotechnology 16: 421) and combinations thereof.

Non-viral delivery systems may also include, but are not limited to,bacterial delivery systems. The use of bacteria as anticancer agents andas delivery agents for anticancer drugs has been reviewed in Expert OpinBiol Ther 2001 March; 1(2):291-300.

Suitable bacteria include, but are not limited to, bacterial pathogensand non-pathogenic commensal bacteria. By way of example, suitablegenera may be selected from Salmonella, Mycobacterium, Yersinia,Shigella, Listeria and Brucella. Recent advances in the pathogenesis andmolecular biology of these bacteria have allowed the rationaldevelopment of new and improved bacterial carriers and more effectivegene expression systems. These advances have improved the performanceand versatility of these delivery systems.

The bacteria may be invasive intracellular bacteria that are able totransfer eukaryotic expression plasmids into mammalian host cells invitro and in vivo. Plasmid transfer may take place when the recombinantbacterium dies within the host cell, either due to metabolic attenuationor induction of autolysis. Alternatively, antibiotics may be used andspontaneous transfer has also been observed, indicating that thisphenomenon ght also occur under physiological conditions. Plasmidtransfer has been reported for Shigella flexneri, Salmonellatyphimurium, S. typhi, Listeria monocytogenes and recombinantEscherichia coli, but other invasive bacteria may also be used.

Bacteria may be used for DNA vaccine delivery. Such bacteria may enterthe host cell cytosol after phagocytosis, for example, Shigella andListeria, or they remain in the phagosomal compartment—such asSalmonella. Both intracellular localisations may be suitable forsuccessful delivery of DNA vaccine vectors.

The bacterial delivery systems may utilise Mycobacterium in the form ofnon pathogenic Mycobacterium strains, genetic transfer systems in theform of cloning and expression vectors, and related technologies toprovide products containing, for example, non toxic immuno-regulatingMycobacterium adjuvants, non toxic immuno-stimulating exogenous antigensspecific for a variety of diseases, and non toxic amounts of cytokinesthat boost the TH-1 pathway (Tunis Med 2001 February; 79(2):65-81).

Salmonella strains—such as attenuated strains—which comprise definedgene deletions, may be used as suitable delivery systems—such as thedelivery of antigens. A number of strategies for delivery by thesestrains have been attempted, ranging from plasmid-based to chromosomalintegration systems. By way of example, Rosenkranz et al. Vaccine 2003,21(7-8), 798-801 describe eukaryotic expression plasmids encodingcytokines, and assessed their capacity to modulate immune responses indifferent experimental models. Plasmids encoding mouse IL-4 and IL-18under cytomegalovirus promoter were constructed and transformed intolive attenuated Salmonella enterica serovar Typhi strain CVD 908-htrA,and Salmonella enterica serovar Typhimurium strain SL3261.

The use of attenuated Salmonella typhimurium as a potential genedelivery vector has been reviewed in Anticancer Res 2002, 22(6A):3261-6.

Brucella abortus may also be used as a suitable delivery system asdescribed by Vemulapalli et al. Infect Immun (2000) 68(6):3290-6.Brucella abortus strain RB51 is a stable, rough, attenuated mutantwidely used as a live vaccine for bovine brucellosis. This strain may beused as a delivery vector, for example, in the delivery of protectiveantigens of other intracellular pathogens to which the induction of astrong Th1 type of immune response is needed for effective protection.

Boyd et al. Eur J Cell Biol (2000) 79 (10) 659-71 describe the use ofYersinia enterocolitica for the delivery of proteins into a wide rangeof cell types. Y. enterocolitica translocates virulence proteins, calledYop effectors, into the cytosol of eukaryotic cells. No limit to therange of eukaryotic cells into which Y. enterocolitica can translocateYops was reported. The Yop effectors YopE, YopH and YopT were eachcytotoxic for the adherent cell types tested, showing that not only isY. enterocolitica not selective in its translocation of particular Yopeffectors into each cell type, but also that the action of these Yopeffectors is not cell type specific. To use the Yersinia translocationsystem for broad applications, a Y. enterocolitica translocation strainand vector for the delivery of heterologous proteins into eukaryoticcells was constructed. This strain and vector combination lacks thetranslocated Yop effectors and allows delivery into eukaryotic cells ofheterologous proteins fused to the minimal N-terminalsecretion/translocation signal of YopE.

U.S. Pat. No. 5,965,381 describes a recombinant Yersinia for thedelivery of proteins into eukaryotic cells. Such Yersinia are deficientin the production of functional effector proteins, but are endowed witha functional secretion and translocation system.

Cell adhesion molecules are a large group of molecules involved in avariety of cell-to-cell and cell-to-extra-cellular matrix (ECM)interactions and are exploited by a number of pathogenic micro-organismsas receptors for cell entry. These molecules may be used for thetargeting and uptake of both gene and drug delivery systems. Celladhesion molecules and their use in gene transfer has been reviewed inAdv Drug Deliv Rev 2000 Nov. 15; 44(2-3):135-52.

The gene gun delivery system may also be used for the delivery of DNA,which is a highly reliable method compared to intramuscular inoculation(Jpn J Pharmacol 2000 July; 83 (3): 167-74).

Viral delivery systems include but are not limited to adenovirusvectors, adeno-associated viral (AAV) vectors, herpes viral vectors,retroviral vectors, lentiviral vectors or baculoviral vectors,venezuelan equine encephalitis virus (VEE), poxviruses such as:canarypox virus (Taylor et al 1995 Vaccine 13:539-549), entomopox virus(L1 Y et al 1998 XII^(th) International Poxvirus Symposium p 144.Abstract), penguine pox (Standard et al. J Gen Virol. 1998 79:1637-46)alphavirus, and alphavirus based DNA vectors.

Examples of retroviruses include but are not limited to: murineleukaemia virus (MLV), human immunodeficiency virus (HIV), equineinfectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV).

A detailed list of retroviruses may be found in Coffin et al(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

Lentiviruses can be divided into primate and non-primate groups.Examples of primate lentiviruses include but are not limited to: thehuman immunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells (Lewis et al 1992 EMBO. J 11: 3053-3058;Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, otherretroviruses—such as MLV—are unable to infect non-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

The vector for use in the present invention may be configured as asplit-intron vector. A split intron vector is described in PCT patentapplications WO 99/15683 and WO 99/15684.

If the features of adenoviruses are combined with the genetic stabilityof retroviruses/lentiviruses then essentially the adenovirus can be usedto transduce target cells to become transient retroviral producer cellsthat could stably infect neighbouring cells. Such retroviral producercells engineered to express 5T4 antigen can be implanted in organismssuch as animals or humans for use in the treatment of angiogenesisand/or cancer.

The vector for use in the present invention may be configured as apsuedotyped vector.

In the design of retroviral vectors it may be desirable to engineerparticles with different target cell specificities to the native virus,to enable the delivery of genetic material to an expanded or alteredrange of cell types. One manner in which to achieve this is byengineering the virus envelope protein to alter its specificity. Anotherapproach is to introduce a heterologous envelope protein into the vectorparticle to replace or add to the native envelope protein of the virus.

The term pseudotyping means incorporating in at least a part of, orsubstituting a part of, or replacing all of, an env gene of a viralgenome with a heterologous env gene, for example an env gene fromanother virus. Pseudotyping is not a new phenomenon and examples may befound in WO 99/61639, WO-A-98/05759, WO-A-98/05754, WO-A-97/17457,WO-A-96/09400, WO-A-91/00047 and Mebatsion et al 1997 Cell 90, 841-847.

Pseudotyping can improve retroviral vector stability and transductionefficiency. A pseudotype of murine leukemia virus packaged withlymphocytic choriomeningitis virus (LCMV) has been described (Miletic etal (1999) J. Virol. 73:6114-6116) and shown to be stable duringultracentrifugation and capable of infecting several cell lines fromdifferent species.

Poxvirus Vectors

Antigens such as TAAs are weakly immunogenic, being recognised as “self”by the immune system and thus tolerated to a large extent. The use ofpoxvirus vectors is sometimes able to cause the antigens to be presentedsuch that this tolerance may be overcome at least in part, (especiallyif immune evasion genes are deleted—see below) thus enabling a host toraise an immune response.

Poxvirus vectors are preferred for use in the present invention. Poxviruses are engineered for recombinant gene expression and for the useas recombinant live vaccines. This entails the use of recombinanttechniques to introduce nucleic acids encoding foreign antigens into thegenome of the pox virus. If the nucleic acid is integrated at a site inthe viral DNA which is non-essential for the life cycle of the virus, itis possible for the newly produced recombinant pox virus to beinfectious, that is to say to infect foreign cells and thus to expressthe integrated DNA sequence. The recombinant pox virus prepared in thisway can be used as live vaccines for the prophylaxis and/or treatment ofpathologic and infectious disease.

Expression of antigen peptide(s) in recombinant pox viruses, such asvaccinia viruses, requires the ligation of vaccinia promoters to thenucleic acid encoding the 5T4 peptide(s). Plasmid vectors (also calledinsertion vectors), have been constructed to insert nucleic acids intovaccinia virus through homologous recombination between the viralsequences flanking the nucleic acid in a donor plasmid and homologoussequence present in the parental virus (Mackett et al 1982 PNAS 79:7415-7419). One type of insertion vector is composed of (a) a vacciniavirus promoter including the transcriptional initiation site; (b)several unique restriction endonuclease cloning sites located downstreamfrom the transcriptional start site for insertion of nucleic acid; (c)nonessential vaccinia virus sequences (such as the Thymidine Kinase (TK)gene) flanking the promoter and cloning sites which direct insertion ofthe nucleic acid into the homologous nonessential region of the virusgenome; and (d) a bacterial origin of replication and antibioticresistance marker for replication and selection in E. Coli. Examples ofsuch vectors are described by Mackett (Mackett et al 1984, J. Virol. 49:857-864).

The isolated plasmid containing the nucleic acid to be inserted istransfected into a cell culture, e.g., chick embryo fibroblasts, alongwith the parental virus, e.g., poxvirus. Recombination betweenhomologous pox DNA in the plasmid and the viral genome respectivelyresults in a recombinant poxvirus modified by the presence of thepromoter-gene construct in its genome, at a site which does not affectvirus viability.

As noted above, the nucleic acid is inserted into a region (insertionregion) in the virus which does not affect virus viability of theresultant recombinant virus. Such regions can be readily identified in avirus by, for example, randomly testing segments of virus DNA forregions that allow recombinant formation without seriously affectingvirus viability of the recombinant. One region that can readily be usedand is present in many viruses is the thymidine kinase (TK) gene. Forexample, the TK gene has been found in all pox virus genomes examined[leporipoxvirus: Upton, et al J. Virology 60:920 (1986) (shope fibromavirus); capripoxvirus: Gershon, et al J. Gen. Virol. 70:525 (1989)(Kenya sheep-1); orthopoxvirus: Weir, et al J. Virol 46:530 (1983)(vaccinia); Esposito, et al Virology 135:561 (1984) (monkeypox andvariola virus); Hruby, et al PNAS, 80:3411 (1983) (vaccinia);Kilpatrick, et al Virology 143:399 (1985) (Yaba monkey tumour virus);avipoxvirus: Binns, et al J. Gen. Virol 69:1275 (1988) (fowlpox); Boyle,et al Virology 156:355 (1987) (fowlpox); Schnitzlein, et al J.Virological Method, 20:341 (1988) (fowlpox, quailpox); entomopox(Lytvyn, et al J. Gen. Virol 73:3235-3240 (1992)].

In vaccinia, in addition to the TK region, other insertion regionsinclude, for example, HindIII M.

In fowlpox, in addition to the TK region, other insertion regionsinclude, for example, BamHI J [Jenkins, et al AIDS Research and HumanRetroviruses 7:991-998 (1991)] the EcoRI-HindIII fragment, BamHIfragment, EcoRV-HindIII fragment, BamHI fragment and the HindIIIfragment set forth in EPO Application No. 0 308 220 A1. [Calvert, et alJ. of Virol 67:3069-3076 (1993); Taylor, et al Vaccine 6:497-503 (1988);Spehner, et al (1990) and Boursnell, et al J. of Gen. Virol 71:621-628(1990)].

In swinepox preferred insertion sites include the thymidine kinase generegion.

A promoter can readily be selected depending on the host and the targetcell type. For example in poxviruses, pox viral promoters should beused, such as the vaccinia 7.5K, or 40K or fowlpox C1. Artificialconstructs containing appropriate pox sequences can also be used.Enhancer elements can also be used in combination to increase the levelof expression. Furthermore, the use of inducible promoters, which arealso well known in the art, are preferred in some embodiments.

Foreign gene expression can be detected by enzymatic or immunologicalassays (for example, immuno-precipitation, radioimmunoassay, orimmunoblotting). Naturally occurring membrane glycoproteins producedfrom recombinant vaccinia infected cells are glycosylated and may betransported to the cell surface. High expressing levels can be obtainedby using strong promoters.

Other requirements for viral vectors for use in vaccines include goodimmunogenicity and safety. MVA is a replication-impaired vaccinia strainwith a good safety record. In most cell types and normal human tissue,MVA does not replicate. Replication of MVA is observed in a fewtransformed cell types such as BHK21 cells. Carroll et al (1997) haveshown that the recombinant MVA is equally as good as conventionalrecombinant vaccinia vectors at generating a protective CD8+ T cellresponse and is an efficacious alternative to the more commonly usedreplication competent vaccinia virus. The vaccinia virus strains derivedfrom MVA, or independently developed strains having the features of MVAwhich make MVA particularly suitable for use in a vaccine, are alsosuitable for use in the present invention.

Preferably, the vector is a vaccinia virus vector such as MVA or NYVAC.Most preferred is the vaccinia strain modified virus ankara (MVA) or astrain derived therefrom. Alternatives to vaccinia vectors includeavipox vectors such as fowlpox or canarypox known as ALVAC and strainsderived therefrom which can infect and express recombinant proteins inhuman cells but are unable to replicate.

In one aspect of the present invention at least one immune evasion geneis deleted from the poxvirus vector.

Viruses, especially large viruses such a poxviruses which have anextensive coding capacity and can thus encode a variety of genes, havedeveloped a number of techniques for evading the immune system of theirhosts. For example, they are able to evade non-specific defences such ascomplement, interferons and the inflammatory response, as well as tointerfere with or block the function of cytokines. A number of theseimmune evasion polypeptides have been deleted from MVA, with theexception of the interferon resistance protein in the left terminalregion.

Poxviruses in general, are large DNA viruses which establish acute,rather than latent, infections. They encode so many antigenic proteinsthat antigenic variation is difficult, thus relying on active immuneevasion to protect themselves from the mammalian immune system. Theypossess a number of genes which encode polypeptides which areresponsible for interfering with a number of aspects of the immunesystem: they disrupt interferon action, interfere with complement,cytokine activity, inflammatory responses and CTL recognition (for areview, Smith et al., (1997) Immunol Rev 159:137-154). Removal of theseproteins is beneficial in promoting the ability of weak immunogensencoded on a poxvirus vector to elicit an immune response in a subject.

An immune evasion gene or polypeptide is a gene, or its product, whichassists the virus in evading the mammalian immune system. Preferably,the gene or gene product interferes with the working of the immunesystem, at least one level. This may be achieved in a number of ways,such as by interfering in signalling pathways by providing competitorsfor signalling molecules, by providing soluble cytokine receptor mimicsand the like.

Immune evasion genes include, but are not limited to, the following:

Interferon evasion genes. Vaccinia possesses at least three genes whichinterfere with IFN action. The E3L gene expresses a 25 Kd polypeptidewhich competes with P1 protein kinase for binding to dsRNA, an eventwhich leads to activation of P1, phosphorylation of eIF2α and resultantfailure of translation initiation complex assembly. This pathway isordinarily responsive to IFN activation, but is impeded by E3Lexpression thus allowing translation initiation to proceed unimpeded.

The K3L gene expresses a 10.5 Kd polypeptide which also interferes withP1 activity, since it is effectively an eIF2α mimic and acts as acompetitor for P1 protein kinase. Its mode of action is thus similar toE3L.

The A18R gene is predicted to encode a helicase, which appears tointerfere with the 2′,5′-oligoadenylate pathway, which is in turn IFNresponsive. 2′,5′-A activates RNAse L, which acts to prevent viraltranslation. Expression of A18R appears to reduce 2′,5′-A levels ininfected cells.

Complement. The product of the B5R gene of vaccinia is known to behighly related to factor H, a regulator of the alternative complementpathway. This pathway may be activated by antigen alone, unlike theclassical pathway. The B5R gene product thus may interfere with thealternative complement pathway.

The C21L gene is in turn related to C4b-binding protein in humans, andinteracts with cells bearing C4b on the surface to prevent binding tothe CR1 complement receptor.

Soluble Cytokine Receptors. The product of the vaccinia WR B15R gene(B16R in Copenhagen strain vaccinia) is related to IL1-R.

The WR gene ORF SalF19R, A53R in Copenhagen strain vaccinia, encodes aTNF receptor. However, in wild-type virus both of these genes arebelieved to be inactive due to fragmentation of the ORFs.

The B8R gene is believed to encode a soluble IFN-γ receptor, providingthe virus with yet another IFN evasion mechanism.

Inflammation. A number of genes are believed to be involved in theprevention of inflammatory responses to viral infection. These includeA44L, K2L, B 13R and B22R.

In one aspect of the present invention, the majority of the immuneevasion genes are deleted from the recombinant poxvirus vector.Preferably, all the immune evasion genes are deleted. Thus, in oneaspect of the present invention, the recombinant poxvirus vector is arecombinant MVA vector in which the K3L interferon resistance proteingene has been disrupted or deleted.

Preferred are poxyiruses which are non-hazardous to the intendedsubject. Thus, for example, for use in humans, poxviruses which areeither host-range restricted, such as avipox viruses, or otherwiseattenuated, such as attenuated strains of vaccinia (including NYVAC andMVA) are preferred. Most preferred are attenuated vaccinia virusstrains, although non-vaccinia strains are usefully employed in subjectswith pre-existing smallpox immunity.

A construct which contains at least one nucleic acid which codes for atumour associated antigen epitope(s) flanked by MVA DNA sequencesadjacent to a naturally occurring deletion, e.g. deletion II, within theMVA genome, is introduced into cells infected with MVA, to allowhomologous recombination.

Once the construct has been introduced into the eukaryotic cell and thetumour associated antigen epitope DNA has recombined with the viral DNA,the desired recombinant vaccinia virus, can be isolated, preferably withthe aid of a marker (Nakano et al Proc. Natl. Acad. Sci. USA 79,1593-1596 [1982], Franke et al Mol. Cell. Biol. 1918-1924 [1985],Chakrabarti et al Mol. Cell. Biol. 3403-3409 [1985], Fathi et alVirology 97-105 [1986]).

The construct to be inserted can be linear or circular. A circular DNAis preferred, especially a plasmid. The construct contains sequencesflanking the left and the right side of a naturally occurring deletion,e.g. deletion II, within the MVA genome (Altenburger, W., Suter, C. P.and Altenburger J. (1989) Arch. Virol. 105, 15-27). The foreign DNAsequence is inserted between the sequences flanking the naturallyoccurring deletion.

For the expression of at least one nucleic acid, it is necessary forregulatory sequences, which are required for the transcription of thenucleic acid to be present upstream of the nucleic acid. Such regulatorysequences are known to those skilled in the art, and includes forexample those of the vaccinia 11 kDa gene as are described inEP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385).

The construct can be introduced into the MVA infected cells bytransfection, for example by means of calcium phosphate precipitation(Graham et al Virol. 52, 456-467 [1973; Wigler et al Cell 777-785 [1979]by means of electroporation (Neumann et al EMBO J. 1, 841-845 [1982]),by microinjection (Graessmann et al Meth. Enzymology 101, 482-492(1983)), by means of liposomes (Straubinger et al Methods in Enzymology101, 512-527 (1983)), by means of spheroplasts (Schaffner, Proc. Natl.Acad. Sci. USA 77, 2163-2167 (1980)) or by other methods known to thoseskilled in the art. Transfection by means of liposomes is preferred.

The recombinant vectors for use in the present invention can have atropism for a specific cell type in the mammal. By way of example, therecombinant vectors of the present invention can be engineered to infectprofessional APCs such as dendritic cells and macrophages. Dendriticcells are known to be orchestrators of a successful immune responseespecially that of a cell mediated response. It has been shown that exvivo treatment of dendritic cells with antigen or viral vectorscontaining such a target antigen, will induce efficacious immuneresponses when infused into syngeneic animals or humans (see Nestle F O,et al. Vaccination of melanoma patients with peptide- or tumorlysate-pulsed dendritic cells, Nat. Med. 1998 March; 4(3):328-32 and KimC J, et al. Dendritic cells infected with poxviruses encodingMART-1/Melan A sensitize T lymphocytes in vitro. J. Immunother. 1997July; 20(4):276-86. The recombinant vectors can also infect tumourcells. Alternatively, the recombinant vectors are able to infect anycell in the mammal.

Other examples of vectors include ex vivo delivery systems, whichinclude but are not limited to DNA transfection methods such aselectroporation, DNA biolistics, lipid-mediated transfection andcompacted DNA-mediated transfection.

The vector may be a plasmid DNA vector. As used herein, “plasmid” refersto discrete elements that are used to introduce heterologous DNA intocells for either expression or replication thereof. Selection and use ofsuch vehicles are well within the skill of the artisan.

Suitably the vector system for administration of the tumour associatedantigen is the TroVax® vaccine, a cancer vaccine in clinical developmentfor delivery of 5T4 using an attenuated vaccinia virus vector (MVA).TroVax® is currently being evaluated in phase II clinical trials in latestage colorectal. renal and prostate cancer patients. Trials usingTroVax are described, for example, in PCT/GB2005/000026. Furthermorevariations or modifications based on TroVax to administer tumourassociated antigens is also envisaged.

Pulsed Cells

The present invention also provides administration of an antigen usingcells pulsed with tumour associated antigen or peptides.

Preferably the cells to be pulsed are capable of expressing MHC class Ior class II molecules.

MHC class I molecules can be expressed on nearly all cell types, butexpression of MHC class II molecules is limited to so-called“professional” antigen presenting cells (APCs); B cells, dendritic cellsand macrophages. However, expression of MHC class II can be induced onother cell types by treating with IFNγ.

Expression of MHC class I or MHC class II molecules can also be achievedby genetic engineering (i.e. provision of a gene encoding the relevantMHC molecule to the cell to be pulsed). This approach has the advantagethat an appropriate MHC haplotype(s) can be chosen which bindspecifically to the peptide(s).

Preferably the cell to be pulsed is an antigen presenting cell, i.e. acell which, in a normal immune response, is capable of processing anantigen and presenting it at the cell surface in conjunction with an MHCmolecule. Antigen presenting cells include B cells, macrophages anddendritic cells. In an especially preferred embodiment, the cell is adendritic cell.

Preferably the cell is capable of expressing an MHC molecule which bindsa peptide according to the first aspect of the invention in its peptidebinding groove. For example, the cell may express one of the followingHLA restriction elements: B8, Cw7 or A2 (for MHC class I).

Peptide pulsing protocols are known in the art (see for exampleRedchenko and Rickinson (1999) J. Virol. 334-342; Nestle et al (1998)Nat. Med. 4 328-332; Tjandrawan et al (1998) J. Immunotherapy 21149-157). For example, in a standard protocol for loading dendriticcells with peptides, cells are incubated with peptide at 50 μg/ml with 3μg/ml β-2 microglobulin for two hours in serum free medium. The unboundpeptide is then washed off.

The pulsed cell of the invention may be used as a vaccine, for exampleto stimulate a prophylactic or therapeutic immune response against aspecific tumour associated antigen.

The present invention therefore also provides a method for treatingand/or preventing a disease which comprises the step of administering apeptide-pulsed cell to a subject in need of same in combination with achemotherapeutic agent.

Nucleic Acid

The antigen for use in the composition or medicament or foradministration in a combination in accordance with the invention may beprovided through a nucleic acid molecule encoding said tumour associatedantigen.

A “nucleic acid”, as referred to herein, may be DNA or RNA,naturally-occurring or synthetic, or any combination thereof. Nucleicacids according to the invention are limited only in that they serve thefunction of encoding a tumour associated antigen peptide in such a waythat it may be translated by the machinery of the cells of a hostorganism. Thus, natural nucleic acids may be modified, for example toincrease the stability thereof. DNA and/or RNA, but especially RNA, maybe modified in order to improve nuclease resistance of the members. Forexample, known modifications for ribonucleotides include 2′-O-methyl,2′-fluoro, 2′—NH₂, and 2′-O-allyl. The modified nucleic acids accordingto the invention may comprise chemical modifications which have beenmade in order to increase the in vivo stability of the nucleic acid,enhance or mediate the delivery thereof, or reduce the clearance ratefrom the body. Examples of such modifications include chemicalsubstitutions at the ribose and/or phosphate and/or base positions of agiven RNA sequence. See, for example, WO 92/03568; U.S. Pat. No.5,118,672; Hobbs et al., (1973) Biochemistry 12:5138; Guschlbauer etal., (1977) Nucleic Acids Res. 4:1933; Schibaharu et al., (1987) NucleicAcids Res. 15:4403; Pieken et al., (1991) Science 253:314, each of whichis specifically incorporated herein by reference.

Nucleic acids encoding suitable antigens or peptide derived therefromwill be familiar to those skilled in the art or can be derived usingmethods which are standard to those skilled in the art. For example, aDNA for use in the present invention is obtainable by chemicalsynthesis, using polymerase chain reaction (PCR) or direct cleavage froma longer polynucleotide, such as the entire tumour associated antigencoding sequence or a fragment thereof.

Nucleic acids encoding suitable antigens and peptides or polyepitopestrings derived therefrom may be codon optimised. Codon optimisation haspreviously been described in WO 99/41397 and WO01/79518. Different cellsdiffer in their usage of particular codons. This codon bias correspondsto a bias in the relative abundance of particular tRNAs in the celltype. By altering the codons in the sequence so that they are tailoredto match with the relative abundance of corresponding tRNAs, it ispossible to increase protein expression. By the same token, it ispossible to decrease expression by deliberately choosing codons forwhich the corresponding tRNAs are known to be rare in the particularcell type. Thus, an additional degree of translational control isavailable. Codon usage tables are known in the art for mammalian cells,as well as for a variety of other organisms.

Variants/Fragments/Homologues/Derivatives

The present invention encompasses the use of nucleotide and amino acidsequences encoding antigens and variants, homologues, derivatives andfragments thereof.

The term “variant” is used to mean a naturally occurring polypeptide ornucleotide sequence which differs from a wild-type sequence.

The term “fragment” indicates that a polypeptide or nucleotide sequencecomprises a fraction of a subject sequence. Preferably the sequencecomprises at least 50%, more preferably at least 65%, more preferably atleast 80%, more preferably at least 90%, most preferably at least 90% ofthe subject sequence. If the frdgment is a fragment of an amino acidthen preferably the fragments are 6-12 amino acids in length. Morepreferably, the fragments are 8, 9 or 10 amino acids in length.

The term “homologue” means an entity having a certain homology with thesubject amino acid sequences and the subject nucleotide sequences. Here,the term “homology” can be equated with “identity”.

In the present context, a homologous sequence is taken to include anamino acid sequence, which may be at least 75, 85 or 90% identical,preferably at least 95 or 98% identical to the subject sequence.Typically, the homologues will comprise the same activity as the subjectamino acid sequence. Although homology can also be considered in termsof similarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

In the present context, a homologous sequence is taken to include anucleotide sequence, which may be at least 75, 85 or 90% identical,preferably at least 95 or 98% identical to the subject sequence.Typically, the homologues will comprise the same activity as the subjectsequence. Although homology can also be considered in terms ofsimilarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

Methods for determining sequence identity are familiar to those skilledin the art.

One suitable homologue of 5T4 is described in GB 0615655.8 hereinincorporated by reference.

Vaccine/Pharmaceutical Composition

The present invention provides a vaccine/pharmaceutical compositioncomprising an antigen, a peptide epitope derived from a tumourassociated antigen, a polyepitope string, a nucleic acid sequence, avector system and/or a cell as described above for use in combinationwith a chemotherapeutic agent or agents.

For administering the antigen or derivatives as described above, thevaccine may by prepared as an injectable, either as liquid solution orsuspension; solid form suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified, or the protein encapsulated in liposomes. The activeimmunogenic ingredients are often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof.

In addition, if desired, the vaccine may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

Further examples of adjuvants and other agents include aluminiumhydroxide, aluminium phosphate, aluminium potassium sulphate (alum),beryllium sulphate, silica, kaolin, carbon, water-in-oil emulsions,oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis,polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A,saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers orother synthetic adjuvants. Such adjuvants are available commerciallyfrom various sources, for example, Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and CompleteAdjuvant (Difco Laboratories, Detroit, Mich.).

Typically, adjuvants such as Amphigen (oil-in-water), Alhydrogel(aluminium hydroxide), or a mixture of Amphigen and Alhydrogel are used.Only aluminium hydroxide is approved for human use.

The proportion of immunogen and adjuvant can be varied over a broadrange so long as both are present in effective amounts. For example,aluminium hydroxide can be present in an amount of about 0.5% of thevaccine mixture (Al₂O₃ basis). Conveniently, the vaccines are formulatedto contain a final concentration of immunogen in the range of from 0.2to 200 μg/ml, preferably 5 to 50 μg/ml, most preferably 15 μg/ml.

After formulation, the vaccine may be incorporated into a sterilecontainer which is then sealed and stored at a low temperature, forexample 4° C., or it may be freeze-dried. Lyophilisation permitslong-term storage in a stabilised form.

The vaccine may be administered in a convenient manner such as by theoral, intravenous (where water soluble), intramuscular, subcutaneous,intranasal, intradermal or suppository routes or implanting (e.g. usingslow release molecules).

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1% to 2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain 10% to 95% of activeingredient, preferably 25% to 70%. Where the vaccine composition islyophilised, the lyophilised material may be reconstituted prior toadministration, e.g. as a suspension. Reconstitution is preferablyeffected in buffer.

Capsules, tablets and pills for oral administration to a patient may beprovided with an enteric coating comprising, for example, Eudragit “S”,Eudragit “L”, cellulose acetate, cellulose acetate phthalate orhydroxypropylmethyl cellulose.

Peptides and polypeptides may be formulated into the vaccine as neutralor salt forms. Pharmaceutically acceptable salts include the acidaddition salts (formed with free amino groups of the peptide) and whichare formed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids such as acetic, oxalic, tartaricand maleic. Salts formed with the free carboxyl groups may also bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine andprocaine.

5T4 peptides may be administered with costimulatory molecules such asthose involved in the interaction between receptor-ligand pairsexpressed on the surface of antigen presenting cells and T cells. Suchcostimulatory molecules can be administered by administration of theprotein molecule or of the corresponding nucleic acid encoding theprotein molecule. Suitable costimulatory molecules include CD40, B7-1,B7-2, CD54, members of the ICAM family (eg ICAM-1, -2, or -3), CD58,SLAM ligands, polypeptides that bind heat stable antigen, polypeptideswhich bind to members or the TNF receptor family (eg 4-1BBL, TRAF-1,TRAF-2, TRAF-3, OX40L, TRAF-5, CD70) and CD 154. Peptides may also beadministered in combination with stimulatory chemokines or cytokinesincluding, for example, IL-2, IL-3, IL4, SCF, IL-6, IL7, IL-12, IL15,IL16, IL18, G-CSF, GM-CSF, IL-1alpha, IL-11, MIP-11, LIF, c-kit ligand,thrombopoietin and flt3 ligand, TNF-α and interferons such as IFN-α orIFN-γ. Chemokines may also be used in combination with the antigen orpeptides, such as CCL3 or CCL5 or may be fused with the peptides of theinvention (eg CXCL10 and CCL7). Where the antigen or peptides areadministered by administering a nucleic acid encoding the peptide, thecostimulatory molecule may also be administered by administering thecorresponding nucleic acid encoding the costimulatory molecule.

For example, treatment with anti-CTLA-4, anti-CD25, anti-CD4, the fusionprotein IL13Ra2-Fc, and combinations thereof (such as anti-CUA-4 andanti-CD25) have been shown to upregulate anti-tumour immune responsesand would be suitable to be used in combination with the peptides of thepresent invention. The regulatory T-cell (Treg) inhibitor ONTAK (IL-2diptheria toxin conjugate DAB₃₈₉IL2) has also been shown to enhancevaccine-mediated antitumour, thus inhibitors of Tregs are also suitablefor use with the vaccines of the present invention.

Heterologous Vaccination Regimes

Regimes for administration of vaccines/pharmaceutic compositionsaccording to the present invention may be determined by conventionalefficacy testing. Especially preferred, however, are regimes whichinclude successive priming and boosting steps. It is observed that suchregimes achieve superior breaking of immune tolerance and induction of Tcell responses (see Schneider et al., 1998 Nat Med 4:397-402) as well asinduction of B cell and antibody responses.

Prime-boost regimes may be homologous (where the same composition isadministered in subsequent doses) or heterologous (where the primimg andboosting compositions are different). For example, the primingcomposition may be a non-viral vector (such as a plasmid) encoding atumour associated antigen and the boosting composition may be a viralvector (such as a poxvirus vector) encoding a tumour associated antigen,wherein either or both of said “tumour associated antigens” is anepitope or polyepitope string of the present invention.

Prophylactic/Therapeutic Methods

The present invention also provides the use of a combination accordingto the present invention in the manufacture of a medicament for use inthe prevention and/or treatment of a disease.

There is also provided a method for treating and/or preventing a diseasein a subject which comprises the step of administering an effectiveamount of a combination according to the present invention.

As used herein, the terms “treatment”, “treating” and “therapy” includecurative effects, palliative effects, alleviation effects, prevention ofprogression, prophylactic effects and any effect which improves thesurvival of a patient.

Where the vaccine is or comprises a class I peptide epitope, the immuneresponse elicited may involve the activation of 5T4 specific cytotoxicT-lymphocytes. Where the vaccine is or comprises a class II epitope, theimmune response elicited may involve the activation of T_(H)1 and/orT_(H)2 cells.

Advantageously, the response is an anti-tumour immunotherapeuticresponse which is effective to inhibit, arrest or reverse thedevelopment of a tumour in a subject.

Chemotherapeutic Agents

“Chemotherapeutic agents” for use in the combination of the presentinvention are those agents which are agents suitable for anti-cancer oranti-tumour therapies.

Suitable chemotherapeutic agents include standard compounds used inchemotherapy, intercalating agents, and platinum containing compounds,for example. Suitable agents include, but are not limited to all-transretinoic acid, Actimide, Azacitidine, Azathioprine, Bleomycin,Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide,Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin,Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine,Hydroxyurea, Idarubicin, Irinotecan, Lenalidomide, Leucovorin,Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone,Oxaliplatin, Paclitaxel, Pemetrexed, Revlimid, Temozolomide, Teniposide,Thioguanine, Valrubicin, Vinblastine, Vincristine, Vindesine andVinorelbine.

In one embodiment, a chemotherapeutic agent for use in the combinationof the present agent may, itself, be a combination of differentchemotherapeutic agents. Suitable combinations include FOLFOX and IFL.FOLFOX is a combination which includes 5-fluorouracil (5-FU),leucovorin, and oxaliplatin. IFL treatment includes irinotecan, 5-FU,and leucovorin.

In one embodiment, the chemotherapeutic agent is cyclophosphamide. Inthis embodiment, the cyclophosphamide may, preferably be administered ina low dose form. Low dose cyclophosphamide has been observed to enhancean antitumour response in an adoptive cell transfer immunotherapyapproach (see Dudley et al. J. Clin. Oncol. (2005) 23, 2346-2357).

In another embodiment, the composition may further comprise a kinaseinhibitor, for separate, simultaneous separate or combined use in thetreatment of tumours. Suitable kinase inhibitors include those whichhave been shown to possess anti-tumour activity (such as gefitinib(Iressa) and erlotinib (Tarceva) and these could be used in combinationwith the peptides. The receptor tyrosine kinase inhibitors, such asSunitinib malate and Sorafenib which have been shown to be effective inthe treatment of renal cell carcinoma are also suitable to be used inthe composition.

Other Combination Therapies

The invention further relates to the use of tumour antigen targetingmolecules, such as anti-tumour antigen antibodies, for example anti-5T4scFvs. Tumour antigen targeting molecules also includes T cell receptors(TCRs), including the synthetic TCRs described in WO 2004/033695 and WO99/60119. These antibodies may be used to (i) to target natural orexogenous 5T4 in situ and/or (ii) deliver immune enhancer molecules,such as B7.1, to natural or exogenous 5T4 in situ (Carroll et al. (1998)J Natl Cancer Inst 90(24):1881-7). This potentiates the immunogenicityof 5T4 in the subject.

The present invention may also be used with the adoptive transfer oftumour infiltrating lymphocytes isolated from patients (Dudley et al. J.Clin. Oncol. (2005) 23:2346-2357).

Diseases

Diseases which can be treated and/or prevented in accordance with theinvention include any of those in which an antigen-specific immuneresponse can contribute to that prevention and/or treatment.

In a preferred embodiment, the disease (which is preventable/treatableusing a combination according to the present invention) is a cancer. Inparticular the disease may be a carcinoma of, for example, the breast,lung, stomach, pancreas, endometrium, cervix, colorectum, kidney orprostate as well as melanoma.

Preferably the disease is one which can be shown to be positive for thetumour associated antigen which is present in the combination.

For example, WO89/07947 describes an immunohistochemical screen ofneoplastic tissues using an anti-5T4 monoclonal antibody. Thus, wherethe tumour associated antigen is 5T4, the disease is, preferably, acancer which can be shown to be 5T4 positive by diagnostic testing (suchas with an anti-5T4 antibody), for example: an invasive carcinoma of theAmpulla of Vater, breast, colon, endometrium, pancreas, or stomach; asquamous carcinoma of the bladder, cervix, lung or oesophagus; atubulovillous adenoma of the colon; a malignant mixed Mullerian tumourof the endometirem; a clear cell carcinoma of the kidney; a lung cancer(large cell undifferentiated, giant cell carcinoma, broncho-alveolarcarcinoma, metastatic leiomyosarcoma); an ovarian cancer (a Brennertumour, cystadenocarcinoma, solid teratoma); a cancer of the testis(seminoma, mature cystic teratoma); a soft tissue fibrosarcoma; ateratoma (anaplastic germ cell tumours); or a trophoblast cancer(choriocarcimoma (e.g. in uterus, lung or brain), tumour of placentalsite, hydatidiform mole).

Dosage and Administration

Administration and CD4⁺CD25⁺ Treg cell count/CD4+CD25+ Treg cellfunction

In some embodiments of the present invention the antigens and vaccinesof the present invention are administered at a reduced CD4⁺CD25⁺ Tregcell count or reduced CD4+CD25+ Treg cell function. Preferably, theoptimum timing of vaccination would co-incide with the period when theCD4⁺CD25⁺ Treg cell count reducing agent or the CD4+CD25+ Treg cellfunction reducing agent, such as a chemotherapeutic agent or agents orOntak, has caused a maximum depletion/reduction of function of Tregs.

Determination of CD4⁺CD25⁺ Treg Cell Count

The degree of depletion can be determined by analysis of peripheralblood mononuclear cells (PBMCs) isolated from patient blood taken at 24h periods following a dose of chemotherapy, until the next dose ofchemotherapy or for up to 6 weeks after the last administration ofchemotherapy. Treg cells are a specific subset of CD4+ T cells thatexpress CD25 at levels higher than CD4 negative cells; and thus Treglevels are assayed by determining the percentage of all CD4+ T cellsthat are CD4+CD25⁺. Thus the levels of CD4+CD25+hi. Therefore the levelsof CD4+CD25+hi (Tregs) T cells relative to total CD4+ T cell levels willbe determined before and after administration the Treg-reducing agent.

In the case of cyclophosphamide, maximum depletion of CD4+CD25+ Tregsoccurred four days after administration of CY (Lutsiak et al. 2005 Blood105:2862-2868).

Levels of other types of T regulatory cells, such as TGF-β producing TH3cells, IL-10 producing Tr1 cells and CD8+CD28− T cells, may bedetermined by secretion of cytokines, staining for some cell surfacemarkers and the ability to suppress immune responses (Marshall et al,2004. Blood. 103:1755-1762; Wei et al, 2005. Cancer Res 65: 5020-6;Leong et al, 2006 Immunol. Lett. 15:229-236; for reviews see Levings andRoncarlo, 2005. CTMI 292:303-326; Huehn, Siegmund and Hamann, 2005. CITR293:89-114; Faria and Weiner, 2005 Immunol. Rev. 206:232-259; Weiner,2001 Immunol. Rev. 182:207-214; Weiner et al, 2001. Microbes infect.3:947-954; Roncarlo et al, 2001 Immunol. Rev. 182: 68-79).

Determination of CD4⁺CD25⁺ Treg Cell Function

Reduction of CD4⁺CD25⁺ Treg cell function will be determined bymeasuring the loss of suppressive activity of these cells in a number ofin vitro assays including proliferation and ELISPOT assays.

Assays can be set up with PBMCs undepleted or depleted of CD4⁺CD25⁺ Tregcells. CD4⁺CD25⁺ Treg cells can be purified from PBMCs, for example byCD4⁺and CD25⁺ separation techniques using magnetic beads or flowcytometry. In the presence of functional CD4⁺CD25⁺ Treg cells, immuneresponses may not be detected due to suppression caused by the Tregs. Inthe absence of functional CD4⁺CD25⁺ Treg cells, immune responses may bedetected. Purified CD4⁺CD25⁺ Treg cells added back to the depleted PBMCswould result in suppression of these immune responses. In the instanceof cells with a reduced function, immune responses may be detected evenwhen said cells are present in the assays. The loss of Treg functionwould be determined by performing such assays on PBMCs taken before andafter administration of the CD4+CD25+ Treg cell function reducing agent.

Chemotherapy Cycles

It is apparent for the skilled person that the compositions, methods anduses of the present invention can be adapted to the specificchemotherapeutic agents and antigens used within the invention withoutundue experimentation. In particular, a chemotherapeutic agent or agentsused in the present invention might require a specific administrationand dosage schedule. These administration and dosage schedules mightvary for different chemotherapeutic agent. Generally, a chemotherapeuticagent or agents might be administered in short frequent intervals (forexample, every 1 or 2 hours) followed by longer periods withoutadministration (for example, 2 week intervals). This succession ofadministration and non-administration constitutes a chemotherapy cycle.A chemotherapy treatment might consist of a number of cycles dependingon the agent used. The period between chemotherapy cycles constitutesthe rest period. A rest period varies between different chemotherapeuticagents and treatments. Examples of such chemotherapeutic agents and therecommended rest periods are given in the table below.

Drug Class Drug Usual Dosage and Route Alkylating drugs Mechlorethamine6 mg/m² IV (nitrogen mustard) Chlorambucil 4-10 mb/day po (Leukeran) 600mg/m²/IV Cyclophosphamide 50-200 mg/m² po (Cytokxan) 1 mg/kg po q 4 wkMelphan (Alkeran) 2-4 g/m²/day Ifosfamide (Ifex) IV × 3-5 days q 3-4 wkAntimetabolites Methotrexate 2.5-5.0 mg/day Folate po antagonist 25-50mg/1 dose/wk po 100-10,000 Mg/m² IV (with rescue) Purine6-Mercaptopurine 100 mg/m²/day antagonist 5-Fluorouracil po Pyrimidine300-1000/m² IV or antagonist continuous infustion Cytarabine 100 mg/m²IV continuous infusion Gemcitabine (Gemzar) 1200 g/m²/wk IV Spindlepoison Vinblastine (Velban 0.1-0.2 mg/kg IV q 7-10 (from days plants)Vincas Vincristine (Oncovin) 1.4 mg/m²/wk IV Vinorelbine (Navelbine) 20mg/m²/wk IV Paclitaxel (Taxol) 135 mg-200 g/mL IV q3 wk Docetaxel(Taxotere) 100 g/m² IV q 3 wk Podophyllotoxins Etoposide (VePesid) 100mg/m²/day IV for 3-5 days 100 mg/day po for 14 days/mo Irinotecan(Camptosar) 100-125 g/m² IV wk IV Topotecan (Hycamtin) 1.5 g/m² IV daily× 5 days q 3-4 wk Antibiotics Doxorubicin 40-75 mg/m² rapidly IV(Adrianmycin) or 30 mg/m/day for 3 days by continuous IV Bleomycin(Blenoxane) 6-15 U/m² sc or IV Mitomycin Usually 10 to 12 mg/m², slowlyIV Nitrosoureas Carmustine (BiCNU) 150-200 mg/m² IV q 6 wk Lomustine(CeeNU) 100-130 mg/m² po q 6 wk Inorganic ions Cisplatin (Platinol)60-100 mg/m² IV or 20 mg/m² IV daily × 5 days Carboplatin (Paraplatin)300 g/m² or target area under the curve of 5-6 IV q 3 wk

It is understood that a treatment, consisting of a number of cycles anddefined rest periods might vary also among different patients.Furthermore, such treatment might vary for different combinations andmixtures of chemotherapeutic agent. A skilled person will select thesuitable number of cycles and rest periods for each patient andchemotherapeutic agent or chemotherapeutic agent combination.

The regimes of administration of antigen and chemotherapeutic agent oragents according to the present invention are adapted to the specificagent and agent combinations used. It is understood that a fulltreatment employing the compositions, methods and uses of the presentinvention could span a number of separate chemotherapy cycles. It isfurther understood that the treatment patterns could be repeated anynumber of times as required. In particular, the compositions, methodsand uses of the present invention cover administration of vaccines priorto and/or, during and/or after administration of a chemotherapeuticagent, a chemotherapeutic cycle or an entire chemotherapeutic treatmentof several cycles in accordance with the chemotherapeutic agent/vaccinecombination chosen. It is further understood that the compositions,methods and uses according to the present invention are used within atreatment regime which includes also other treatments such as surgeryand/or radiotherapy.

Preferably the antigen would be administered during rest periods withinor after chemotherapy cycles. Timing could be optimised by measuring thereduction of Treg levels or Treg function during each chemotherapeutictreatment.

Dosage

A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will depend on a variety of factors includingthe activity of the specific compound employed, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and theindividual undergoing therapy. The dosages disclosed herein areexemplary of the average case. There can of course be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

For example, suitable doses for the administration of thechemotherapeutic regimes FOLFOX and IFL are set out in the examplessection herein. In addition, suitable regimes for the administration ofthese regimes in combination with administration of a tumour antigen areset out herein.

The invention is further described, for the purposes of illustrationonly, in the following examples in which reference is made to thefollowing Figures.

FIG. 1 shows a diagrammatic of the vaccination and monitoring regimedescribed in the Examples section.

FIG. 2: TV2-FOLFOX: Tumour dimensions throughout the clinical trial timecourse. The figure illustrates the sum of the target tumour lesions forevaluable patients at 3 CT scan time points (prior to TroVax vaccination(screen) and at weeks 14 and X+8).

FIG. 3 TV2-IFL: Tumour dimensions throughout the clinical trial timecourse. The figure illustrates the sum of the target tumour lesions at 3time points: prior to TroVax vaccination (screen) and at weeks X andX+14.

FIG. 4 a shows 5T4 Responses in TV2-IFL, FIG. 4 b shows 5T4 Responses inTV2-FOLFOX, FIG. 4 c shows MVA Responses in TV2-IFL, FIG. 4 d shows MVAResponses in TV2-FOLFOX, FIG. 4 e shows TT Responses in TV2-IFL and FIG.4 f shows TT Responses in TV2-FOLFOX

FIG. 5 Percentages of CD4⁺CD25⁺ Tregs (right hand quadrant) for patientTV2-016 at various time points during the TV2 trial. Results are shownas the percentage of CD4⁺ T cells.

FIG. 6 Confirmation of CD4⁺ CD25⁺ Treg phenotype by staining forintracellular FoxP3 expression. CD4⁺ T cells were gated (A) and theirCD25 expression ascertained (B). CD25^(+hi) expressers were selected bysetting a strict quadrant (B; top right quadrant). The cells in thisright hand quadrant were gated and most expressed FoxP3 (C; top righthand quadrant).

FIG. 7. The mean percentage of CD4⁺CD25⁺ Tregs within the CD4⁺ T cellpopulation for TV2-IFL (black n=7) and −FOLFOX (grey n=6) patients.

EXAMPLES Summary

These examples summarise the kinetics of immune responses detected inboth TV2 IFL and FOLFOX trials and investigates relationships betweenclinical and immunological responses.

Immune Responses and Kinetics

All evaluable patients in both TV2 IFL (n=12) and FOLFOX (n=11) clinicaltrials mounted 5T4 specific cellular and/or humoral immune responses asdetailed below:

Trial Nos patients showing 5T4 (Nos. evaluable specific immune responsespatients) ELISA Proliferation ELISPOT Any Assay IFL (n = 12) 10 (83%) 10(83%) 11 (92%) 12 (100%) FOLFOX (n = 11) 10 (91%) 10 (91%) 10 (91%) 11(100%)

-   -   In both trials, mean antibody titres increased following each of        the 6 vaccinations (compared to the previous sampling time        point).    -   In the FOLFOX trial, mean antibody titres during chemotherapy        (weeks 4-19) were comparable to post-chemotherapy (weeks        X+2-X+10). However, in the IFL trial, mean antibody titres        during chemotherapy were significantly lower than        post-chemotherapy suggesting that the IFL chemotherapy regimen        may effect on antibody responses.    -   In both trials, the greatest mean antibody titre occurred at        week X+8 i.e. following the 6^(th) immunisation.    -   Mean 5T4 proliferative responses showed little or no increase        following any of the 6 vaccinations, although individual        patients did show increases.    -   In both trials, proliferative responses were greatest following        completion of chemotherapy compared to during chemotherapy.    -   In both trials, 5T4 specific proliferative responses showed a        significant increase between weeks 19 and X+2; this was not the        case for MVA or TT responses suggesting that both chemotherapy        regimes impact on the detection of proliferative responses to a        self antigen but not to foreign antigens ex vivo.    -   In general, patients enrolled into the TV2 trials mounted 5T4        specific cellular and humoral immune responses of greater        magnitude and longevity than seen in an earlier trial (TV1) in        which patients recruited onto a TroVax® monotherapy trial had        been previously treated with chemotherapy some time before        TroVax was administered.

Clinical Responses

Possible trends relating 5T4 specific immune responses with clinicalresponse were investigated by ranking the magnitude of each patients'immune response alongside their reported tumour response.

-   -   TV2-FOLFOX: The data presented indicates that high 5T4 specific        antibody titres and ELISPOT (but not proliferative) responses        are associated more frequently with complete and partial        responses than with stable or progressive disease.

This observation is consistent when responses are analysed over theentire monitoring timecourse, during and post chemotherapy. If data fromboth ELISA and ELISPOT are integrated and the magnitude of the combinedresponses tabulated alongside clinical responses, higher scores clustermore frequently with positive tumour responses (CR+PR). Patientsclassified as SD or PD at 14wk or X+8wk have the lowest combined scores.Combining ELISA and ELISPOT data yields stronger trends than each assayanalysed individually.

-   -   TV2-IFL: Strong immune responses in the IFL trial occurred        primarily following completion of chemotherapy. Clinical benefit        will be assessed by monitoring survival and making comparisons        between immunological responses and overall survival will be        investigated once the data are available.

Data Analysis: Analysis of Immune Response Kinetics

-   a. Within each trial, only data from evaluable (per protocol)    patients have been analysed.-   b. All data are presented in a similar manner. Antigen specific    responses are tabulated for each evaluable patient at each time    point analysed. In addition, the mean antigen-specific immune    responses are tabulated on a population basis for each trial at:    -   1. EACH time point    -   2. Prior to vaccination (weeks −2 and 0)    -   3. During chemotherapy (weeks 4-19)    -   4. Post-chemotherapy (weeks X+2 to X+14 (IFL) or X+10 (FOLFOX))    -   It should be stated that 2 TroVax injections occur during        chemotherapy and 2 following completion of chemotherapy.        Furthermore, the 15 week time period during chemo includes 6        blood sampling time points. Following completion of        chemotherapy, IFL patients are monitored for 14 weeks which also        includes 6 sampling time points. FOLFOX patients are monitored        for 10 weeks following completion of chemotherapy and this        comprises 5 sampling time points.-   c. The FOLD increase in immune response after each vaccination is    tabulated. In addition, the fold increase between weeks 19 and X+2    (i.e. the end of chemotherapy and following completion of    chemotherapy) is also noted.

Data Analysis: Analysis of Correlates Between Immune Response and TumourResponse

The MEAN 5T4 specific immune responses have been determined for allevaluable patients for each of the 3 core immuno-monitoring assays(ELISA, proliferation and ELISPOT). Responses have been ranked inascending order and compared against the respective tumour responses(some CT scans are outstanding). Mean 5T4 specific immune responses weredetermined over 3 time periods: (i) Over all post TroVax time pointsi.e. from week 2 to week X+10 for FOLFOX patients and week 2 to X+14 forIFL patients, (ii) Over all post-TroVax time points occurring during theperiod of chemotherapy (weeks 4-19) and (iii) over all post-TroVax timepoints occurring following completion of chemotherapy (weeks X+2 to X+10for FOLFOX or X+14 for IFL).

Furthermore, 5T4 specific ELISPOT, proliferation and antibody responseswere integrated and compared against the reported clinical responses.

To achieve this, each evaluable patient was simply scored relative tothe magnitude of the greatest immune response detected. For example, thepatient with the highest mean 5T4 antibody titre across the selectedmonitoring period (i.e. all post TroVax time points, during chemo, postchemo) was given a score of 100%, all other patients were scored as apercentage of the greatest response. To integrate the assays, the scoresper assay for each patient were simply summed. Such an analysis doestake into consideration the magnitude of the patients' immune responsesbut gives equal “weight” to all assays.

Methods and Protocols Trovax+FOLFOX

A phase II clinical trial for an open label study of TroVax given inconjunction with 5-fluorouracil/leukovorin/oxaliplatin (FOLFOX) wasconducted for determining safety and immunogenicity before, during andafter chemotherapy.

a) Study Design

This is an open label administration of up to six injections of TroVaxin conjunction with a standard schedule of 5-FU/oxaliplatin/leukovorinin patients with advanced colorectal cancer. A total of fifteen patientsare enrolled in order to obtain 10 evaluable patients. The dosageregimen is two injections, given before chemotherapy, two duringchemotherapy (assumed to last for up to 6 months) and two injectionsafter chemotherapy has ceased. Patients could be on study for a maximumof 40 weeks to assess tolerability of the treatment regimen, inductionof humoural and cellular immunity to 5T4 cell surface antigen, andimmune response to the vector

b) Treatment Trovax

Patients are immunised with a single intramuscular injection of Trovax10× at weeks 0, 2, 11, 17, and 2 and 6 weeks after last dose ofchemotherapy

Oxaliplatin/5-FU/Leukovorin

Patients commence chemotherapy at week 4. The chemotherapy is given at 2weekly intervals for 12 weeks (6 cycles, weeks 4, 6, 8, 10, 12, 14). Anassessment of response is made in the week after completion of cycle 6(week 14; CT/MRI scan). Further chemotherapy cycles (up to a maximum of6 cycles, weeks 16, 18, 20, 22, 24, 26) are administered if deemed inthe patient's interest by the investigator.

c) Concurrent Treatments

The 5-FU/leukovorin chemotherapy is given by the modified de Gramontregimen (MdG) along with oxaliplatin, (OxMdG).

The standard regimen given at the Leeds Teaching Hospitals NHS Trust isas follows;

-   1 Two hour infusion of leukovorin 175 mg (or 350 mg of racemic    folinic acid) in 250 ml 5% dextrose.    -   2 Two hour infusion of oxaliplatin 85 mg/m² in 250 ml 5%        dextrose.    -   3 5 minute bolus injection of 5-fluorouracil 400 mg/m².    -   4 46 hour infusion of 5-fluorouracil 2400 mg/m² given through a        Baxter LV5 pump.

Antiemetics, including intravenous 5HT-3 antagonists and dexamethasone,are given prior to each cycle of chemotherapy. Oral antiemetic therapy,consisting of a three-day course of reducing doses of dexamethasone anddomperidone, as required, is given to the patients. All antiemetictherapy required is recorded in the CRF.

TroVax+IFL a) Treatment Plan and Methods

Patients complying with the entry criteria are invited to enter thestudy. After giving fully informed consent, patients are subjected to aphysical examination to document general fitness to proceed with thetrial. If possible, tissue is obtained for determination of 5T4 status.At that time, metastases and/or local recurrence are documented usingrelevant CT scans and the level of CEA (5 ml) surface antigen in bloodis checked. Blood is drawn for haematology and clinical chemistry (10ml), pituitary hormone screen (ACTH, TSH, LH, FSH; 10 ml),auto-antibodies (10 ml) and immunological testing (antibodies to 5T4 andvector, cellular responses to 5T4 and other antigens; 100 ml). Thisscreen should not occur more than two weeks before the immunisationschedule begins.

At week 0 and week 2, the patients are immunised with a singleintramuscular injection of TroVax. Before both injections, blood isagain drawn for measurement of CEA and immunological testing (100 ml).

Patients commence chemotherapy at week 4. The chemotherapy continues attwo weekly intervals for up to 12 months if the patients respond,although six months is the common duration of treatment. Blood isobtained prior to the dose of chemotherapy at weeks 4 and 6 for CEA andimmunological testing (100 ml). Blood is obtained for haematology (5 ml)before each dose of chemotherapy.

Further injections with TroVax are given at weeks 11 and 17 (i.e. in theinterval between doses of chemotherapy. Blood is drawn for CEA andimmunological testing (100 ml) before each of these injections and twoweeks later (weeks 13 and 19).

The chemotherapy then continues for as long as is decided appropriate bythe patient's physician. At the end of chemotherapy, CT scan restagingof the disease are obtained. Two weeks after the chemotherapy ends (X+2weeks), TroVax is again given by a single intramuscular injection.Before the injection and 2 weeks after that (X+4 weeks), blood isobtained for CEA and immunological testing (100 ml). A final injectionis given four weeks after the previous injection (X+6 weeks). Beforethis injection and two (X+8 weeks) and four weeks (X+10 weeks) afterthat, blood is obtained for CEA and immunological testing (100 ml). Afinal follow up visit is conducted four weeks later (X+14 weeks).

b) Concurrent Treatments

De Gramont regimen: Patients receive leucovorin (200 mg/m²/day) as atwo-hour intravenous infusion, followed by 5-FU as an intravenous bolusat 400 mg/m²/day, and then as a 22-hour continuous infusion at 600mg/m²/day, repeated on 2 consecutive days. Irinotecan (180 mg/m²;30-minute intravenous infusion) is administered on day 1, simultaneouslywith leucovorin administration. This cycle is repeated every 2 weeks.

Modified De Gramont: Patients receive leucovorin and bolus 5FU on day 1,followed by infusional 5FU over 46 hours.

Patients included in this trial may not be given any other anticancertreatments for the duration of the trial. A requirement for suchtreatment necessitates removal of the patient from the trial.

Medications intended to relieve symptoms are prescribed at thediscretion of the Investigator and recorded in the Case Report Form(CRF). The patients should also keep a record of any over the countermedicines consumed and these should be noted in the CRF.

Immunomonitoring

The assays used to undertake the immuno-monitoring can be split intothose which measure either cellular or humoral responses and are listedbelow:

Measurement of Cellular Responses 1. Proliferation Assay

This assay is performed using PBMCs processed from fresh blood and setup on the day of blood sampling.

The proliferation assay is based upon the ability of immune cells(primarily CD4⁺ T cells) to respond to specific proteins. One of theways in which a T cell will respond following interaction with itstarget protein, is by rapid cell division. The response to such stimulican simply be measured by counting the number of cells present in aculture before and after the addition of a stimulating agent. However,this can be both laborious and difficult since the responding cells mayconstitute only a small proportion of the total cell population. Inpractice therefore, any enhanced cell division is measured by theincorporation of ³H-Thymidine into cellular DNA, a process which isclosely related to underlying changes in cell number. The proliferativeresponses induced by a test protein can be compared to that induced bymedium alone (no stimulation control). The data are transformed to yielda stimulation index (S.I.) which is defined as: “Mean CPM of PBMCsincubated with test antigen divided by the mean CPM of PBMCs incubatedwith medium alone”. This relative measure of cellular activity is widelyutilised to enable comparisons between samples taken during longitudinalstudies (e.g. patients enrolled in clinical trials) and to handledifferences in the background proliferation to medium alone.

Preparation of plasma and PBMCs from whole blood and measurement ofantigen specific proliferative responses in clinical samples wereaccording to standard techniques using a cell harvester and TopCount.Methods are described, for example, in Braybrooke J P, Slade A,Deplanque G, et al: Phase I study of MetXia-P450 gene therapy and oralcyclophosphamide for patients with advanced breast cancer or melanoma.Clin Cancer Res 11:1512-1520, 2005 and Harrop R, Ryan M G, Golding H, etal: Monitoring of human immunological responses to vaccinia virus.Methods Mol Biol 269:243-266, 2004.

2. ELISPOT

The enzyme linked immunospot (ELISPOT) assay was described more than 13years ago for the detection of specific immune cells at the single celllevel. The ELISPOT assay is used for a wide range of applicationsincluding the monitoring of cellular responses in patients with cancerundergoing immunotherapeutic treatment. The IFNγ ELISPOT assay exhibitsa high level of sensitivity that permits detection of <10 respondingcells per million PBMCs. With this assay, it is possible to detectmemory T cells functionally responding to an antigenic stimulus throughthe secretion of the cytokine IFNγ. Furthermore, the IFNγ ELISPOT assaydoes not require the use of fresh cells or radioactive substances,making it a simpler and more transferable technique than other assayssuch as the Chromium release assay which has been traditionally used invaccine trials to measure T cells responses.

An important advantage of the IFNγ ELISPOT is that it is a directmeasurement of a Th1 cell-mediated immune response. As such, it isuseful for monitoring the effectiveness of a vaccine to inducecell-mediated immunity. In addition, freshly collected or frozen PBMCscan be used for evaluation in the IFNγ ELISPOT. This is a distinctadvantage in the analysis of samples taken from patients at multipletimepoints throughout a clinical trial program. The use of frozen PBMCsenables the batching of samples and hence reduces the overallvariability of the assay. In addition, the availability of frozen testsamples means that assays can be repeated.

PBMCs are plated into coated wells and incubated overnight at 37° C., 5%CO₂ with the appropriate antigen. After approximately 6 hours ofco-culture, memory cells specific for the antigen begin to secrete IFNγthat is, in turn, captured by the membrane bound antibody. Thus, IFNγbinding occurs in the immediate environment surrounding thecytokine-secreting cell. After approximately 20 hours, the cells arewashed off. Subsequently, the assay utilises two high affinitycytokine-specific antibodies directed against different epitopes on thesame cytokine molecule. Spots are generated with a colourimetricreaction in which soluble substrate is cleaved, leaving an insolubleprecipitate at the site of the reaction. The resulting spot represents afootprint of the original cytokine producing cell. The number of spotsis a direct measurement of the frequency of cytokine producing T cellsand the number of spots increases proportionately with the strength ofthe immune response. Activated CD8⁺ T Cells, CD4⁺helper T cells and NKcells secrete this cytokine. Comparison of the frequency ofantigen-specific T cells before, during and after an immunisation cycleshould reflect the relative immunogenicity of the vaccine beingevaluated.

Preparation of plasma and PBMCs from whole blood and measurement ofcellular immune responses to vectors in clinical trial samples wereperformed by ELISPOT using an automated ELISPOT plate reader followingstandard protocols as described, for example, in Braybrooke J P, SladeA, Deplanque G, et al: Phase I study of MetXia-P450 gene therapy andoral cyclophosphamide for patients with advanced breast cancer ormelanoma. Clin Cancer Res 11:1512-1520, 2005. Harrop R, Ryan M G,Golding H, et al: Monitoring of human immunological responses tovaccinia virus. Methods Mol Biol 269:243-266, 2004.

Measurement of Humoral Responses (ELISA)

The enzyme linked immunosorbent assay (ELISA) is based upon the abilityof antibodies to bind their target protein in a highly specific manner.The analysis of antigen-specific antibody responses by ELISA is a widelyutilised and well-established technique. The assay can be used toprovide a relative measure of antigen-specific antibody concentrationsin serum (or other fluids). One of the many applications of thetechnique is to monitor antibody levels following vaccination todetermine whether treatment increases the concentration of the antibodyof interest.

To measure the antigen specific antibody response, the target protein isbound to the surface of a 96-well ELISA plate. Following a blocking step(to minimise non-specific binding of antibodies directly to theplastic), plasma is added to the plate and incubated at roomtemperature. Each well is then washed to further minimise non-specificbinding of antibodies. A secondary antibody, specific for the species ofthe test serum (e.g. anti-human), is added to each well. The secondaryantibody is conjugated to an enzyme (e.g. peroxidase) which, uponincubation with a chromogenic substrate (e.g. OPD) catalyzes itsconversion into a coloured, soluble product which can be quantifiedusing a spectrophotometer. Generally, the greater the colour change thegreater the concentration of antibody present in the test serum

Preparation of plasma and PBMCs from whole blood and measurement ofantigen specific antibody titres were performed by ELISA followingstandard techniques and using a Dynex MRX plate reader for immunologicalassays along with a Dynex plate washer in accordance with themanufacturers instructions. Suitable methods are described, for example,in Braybrooke J P, Slade A, Deplanque G, et al: Phase I study ofMetXia-P450 gene therapy and oral cyclophosphamide for patients withadvanced breast cancer or melanoma. Clin Cancer Res 11:1512-1520, 2005.Harrop R, Ryan M G, Golding H, et al: Monitoring of human immunologicalresponses to vaccinia virus. Methods Mol Biol 269:243-266, 2004.

Assay Controls

For the measurement of antibody responses by ELISA, pooled human plasmarecovered from 5 healthy donors is used as a negative control. As apositive control, plasma recovered from patients enrolled in previousclinical trials (patient TV1-102 (10wk) or patient BC2-102 from TV1 orBC2 clinical trials respectively) is used. Both negative and positivecontrol plasma are included on all assay plates and “pass/fail”acceptance criteria applied to each plate.

Acceptance Criteria/Statistical Methods to be Used.

Assay acceptance criteria and data handling are followed. A positiveimmune response due to vaccination is defined separately for each assayas detailed below:

ELISA

Antibody titre is defined as the greatest dilution of plasma at whichthe mean optical density (O.D.) of the test plasma is ≧2 fold the meanO.D. of the negative control (normal human serum; NHS) at the samedilution.

A positive 5T4 specific antibody response induced by vaccination will bereported if:

-   -   a. The antibody titre compared to NHS is ≧10 and    -   b. The post-injection antibody titre is ≧2 fold the antibody        titre determined at both of the pre-injection time-points.

Proliferation Assay

Results from proliferation assays will be reported as a stimulationindex (S.I.) which is defined as:

S.I.=Incorporation of ³H-Thymidine by PBMCs cultured with testantigen/Incorporation of ³H-Thymidine by PBMCs cultured with mediumalone

A positive 5T4 proliferative response induced by vaccination will bereported if:

-   -   a. The stimulation index (S.I.) to the 5T4 antigen (protein or        peptide) is ≧2 and    -   b. The C.V. of replicates containing the 5T4 antigen is <100%        and    -   c. The S.I. induced by the 5T4 antigen after immunisation is ≧2        fold greater than the        -   highest S.I. induced by the antigen at either of the            pre-injection time-points.

ELISPOT

A positive 5T4 ELISPOT response induced by vaccination will be reportedif:

-   -   a. The mean spot forming units (SFU)/well in response to a 5T4        antigen (protein or peptide) is ≧3 fold the mean SFU/well in        wells containing medium alone and    -   b. The mean SFU/well in response to a 5T4 antigen is ≧10 and    -   c. The 5T4 antigen specific precursor frequency (number of        antigen specific cells per 10⁶ total PBMCs), after immunisation        is ≧2 fold the precursor frequency at either of the        pre-injection time-points.

Results 1. TV2-FOLFOX 1.1 Source Clinical and Patient Data

Seventeen patients were recruited into the TV2-FOLFOX trial of whom 11became evaluable for assessment of immunological responses (Table 1).

TABLE 1 Clinical Data.

All 17 ITT (intention to treat) patients are included in the table.Evaluable patients (n = 11) are indicated by shading (survival datacurrent to date). Key: WD = Patient withdrawn therefore no resultavailable; N/A = Measurement taken but not currently available.

FIG. 2 shows TV2-FOLFOX: Tumour dimensions throughout the clinical trialtime course. The figure illustrates the sum of the target tumour lesionsfor evaluable patients at 3 CT scan time points (prior to TroVaxvaccination (screen) and at weeks 14 and X+8).

Of the 11 evaluable patients, all showed a reduction in tumour burden atweek 14. CT scans are only available for 4 patients at week X+8, all ofwhich showed further decreases in tumour load compared to week 14.

1.2 Immunological Responses 1.2.1 Antibody Responses

5T4 specific antibody titres for each evaluable patient across theentire monitoring time course are illustrated in table 2.

TABLE 2 Table 2: 5T4 specific antibody responses. Results are expressedas a 5T4 specific antibody titre (the greatest serum dilution at whichthe test sample has a mean O.D. (490 nm) ≧ 2 fold that of the negativecontrol sample (normal human serum; NHS)) at each sampling time point.Results tabulated in bold which have ≧10 represent positive antibodyresponses. Mean titres are reported for all patients at each time pointand also at multiple time points pre-vaccination, during chemotherapyand post chemo. In addition, the fold increase in mean antibody titrecompared to the previous time point is shown after each vaccination.Patient 5T4 Specific Antibody Titre at Timepoints (weeks) Post PrimaryImmunisation No −2 0 2 4 6 11 13 17 19 X + 2 X + 4 X + 6 X + 8 X + 10101 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 102 <10 <1010 1280 80 40 40 20 40 20 80 80 80 80 103 <10 <10 <10 80 20 <10 <10 <1020 <10 20 20 320 320 104 <10 <10 <10 <10 <10 <10 10 <10 10 <10 <10 <1040 20 105 <10 <10 <10 40 10 <10 <10 <10 <10 <10 <10 <10 160 80 107 <10<10 <10 160 1280 160 160 80 320 <10 160 160 1280 160 108 <10 <10 <10 4040 <10 1280 320 320 20 640 160 640 1280 113 <10 <10 <10 160 <10 <10 20<10 <10 <10 <10 <10 <10 <10 114 <10 <10 20 320 160 <10 <10 <10 <10 <10<10 <10 <10 10 116 <10 <10 10 20 20 <10 10 <10 80 <10 80 40 80 40 117<10 <10 <10 10 10 <10 10 <10 40 <10 20 <10 160 80 Mean <10 <10 3.6 207147.3 18.2 139.1 38.2 75.5 3.6 90.9 42 250.9 188.2 Titre Fold ↑ 52 7.7 20.05 23 3.7 Mean <10 3.6 102.6 115.1 Titre

Over the entire post-TroVax immuno-monitoring time course, the mean 5T4specific antibody titre was 100. The majority of patients sero-convertedby week 4 (i.e. following 2 TroVax vaccinations). The mean antibodytitres in the FOLFOX trial were high during chemotherapy (mean titre of103), decreased following the completion of chemotherapy (between weeks19 and X+2) and increased moderately following completion ofchemotherapy (mean titre of 115). Increases in the mean antibody titrewere seen after each of the 6 vaccinations.

1.2.2 Proliferative Responses

Tables 3 a-b detail antigen specific proliferative responses for eachevaluable patient across the entire monitoring time course. Theresponses to 5T4 (3a) and MVA (3b) have been analysed. 5T4 representsthe key antigen against which it is hoped to induce a potent andlong-lived immune response. As the vector used to deliver 5T4, MVArepresents a very useful “internal control” on which responses can be“benchmarked”. Patients are expected to mount strong immune responsesagainst MVA (a complex and foreign pathogen) and such responses can becompared, both in terms of their magnitude and kinetics, to thoseinduced against 5T4.

Tables 3a-b: Summary proliferative responses of PBMCs recovered frompatients following in vitro restimulation with 5T4 (a) and MVA (b).Results are expressed as a stimulation index (proliferation induced bymedium alone÷proliferation induced by test antigen). A stimulation index≧2 (indicated by bold text) is considered to be a positive response atthat time point. Proliferative responses which are positive (S.I.≧2) andat least 2 fold greater than the pre-injection (−2 or 0 week) responsesare indicated in bold.

TABLE 3a TV2-FOLFOX: Proliferative responses to 5T4 Sampling Time points(weeks) Patient −2 0 2 4 6 11 13 17 19 X + 2 X + 4 X + 6 X + 8 X + 10101 23.4 9.4 12 3.5 17 29.1 2 1.7 1.9 4.4 1.9 0.8 2.7 1.1 102 10.4 2.90.3 2.1 4.4 11 20.7 5.2 0.72 2.45 6.05 1.99 6.97 6.21 103 4.7 2.1 2.56.3 6.2 1.8 0.6 2.7 2.05 3.31 7.25 4.86 2.15 2.57 104 1.6 0.6 0.6 0.4 11.16 0.64 1.13 0.41 7.11 2.90 1.10 16.10 0.24 105 2.3 0.9 1.6 3.4 1.20.9 1.2 2.41 1.33 8.67 9.75 13.53 17.34 0.60 107 2.6 1 1.3 1.5 0.96 0.537.27 4.49 1.78 7.60 13.63 24.55 16.47 26.93 108 0.7 3.8 0.3 0.90 5.020.41 2.57 5.00 7.85 1.46 6.35 1.39 11.93 5.39 113 0.57 2.11 1.25 0.759.95 5.33 9.52 4.19 3.54 2.23 8.48 12.66 7.91 52.68 114 3.50 1.71 0.392.11 1.08 4.26 1.66 0.81 4.09 9.18 26.66 5.76 10.17 51.99 116 0.38 0.3911.41 4.21 5.92 9.71 0.66 5.84 3.10 23.67 7.36 11.56 0.78 18.66 117 2.651.52 1.68 6.86 9.93 8.38 0.65 2.09 3.95 5.57 12.71 18.35 25.99 19.53Mean 4.8 2.4 3 2.9 5.7 6.6 4.3 3.2 2.8 6.9 9.4 8.8 10.8 16.9 SI Fold ↑1.2 0.97 0.7 0.9 2.5 1.4 1.2 Mean 3.6 4.3 10.5 SI

TABLE 3B TV2-FOLFOX: Proliferative responses to MVA Sampling Time points(weeks) Patient −2 0 2 4 6 11 13 17 19 X + 2 X + 4 X + 6 X + 8 X + 10101 4.7 2.8 69 26.7 23 7.8 2.7 1.1 15.6 3.7 0.7 1.1 2.0 14.2 102 1.5 1.36.3 6 3.2 5.6 6.5 5 4.19 22.76 10.68 1.98 33.17 16.06 103 0.7 0.9 13.85.8 0.3 7.5 6.3 14.7 72.1 5.96 17.92 17.93 5.35 12.01 104 0.4 0.9 5.40.1 2.6 0.4 0.56 11.33 2.40 1.92 1.47 0.92 17.21 0.62 105 1.3 0.5 1 45.44.8 2.58 94.32 9.06 22.51 46.25 143.03 80.49 116.15 0.80 107 1.4 0.2 762.1 45.76 39.37 71.10 43.13 1.13 30.23 351.08 131.36 118.01 167.20 1080.4 0.4 19.5 18.12 3.11 33.92 1.27 40.55 25.41 3.00 155.15 3.22 154.929.90 113 21.64 18.07 40.50 18.74 8.92 6.10 15.94 8.22 4.71 2.59 37.3551.55 40.05 69.76 114 0.50 1.29 1.17 11.21 1.12 2.38 20.86 1.62 5.4526.36 201.10 51.28 39.54 101.36 116 0.66 0.31 37.81 14.96 6.15 4.61 1.314.65 2.06 13.86 4.83 2.15 0.74 3.65 117 2.83 2.13 13.34 170.57 48.1023.02 0.63 10.37 11.70 34.63 164.26 122.77 274.13 236.88 Mean 3.3 2.619.5 34.5 13.4 12.1 20.1 13.6 15.2 17.4 98.9 42.2 72.8 57.5 SI Fold ↑7.5 1.8 1.7 1.1 1.1 5.7 1.7 Mean 2.9 18.2 57.8 SI

The mean proliferative responses to 5T4 and MVA show differences in boththe magnitude and longevity of the response. Each antigen will beaddressed in turn:

A. 5T4

For all evaluable FOLFOX patients, the mean 5T4 specific proliferativeresponse was elevated following TroVax vaccination compared topre-injection (6.8 compared to 3.6). Such responses were strongerfollowing completion of chemotherapy compared to during chemotherapy(10.5 compared to 4.3 respectively). This differential is most markedbetween weeks 19 and X+2 i.e. between the last time point at which mostpatients were still receiving chemotherapy and the first time pointfollowing completion of chemotherapy. This observation was particularlyunexpected because patients did not receive a vaccination within thisperiod (the last vaccination was at week 17). FOLFOX patients showed a2.5 fold increase in mean proliferative responses between weeks 19 andX+2, which was the greatest fold increase observed between consecutivetime points, being greater than following any of the 6 vaccinations.Very small increases in the mean 5T4 proliferative responses weredetected in the FOLFOX trial following the 1^(st), 5^(th) and 6^(th)vaccinations but not at vaccinations given during chemotherapy.

B. MVA

As expected, the mean proliferative responses to MVA were of greatermagnitude than the corresponding 5T4 responses. The mean MVA specificproliferative response following TroVax vaccination was 35 compared to apre-injection SI of 2.9. The MVA specific proliferative responses weregreater following completion of chemotherapy than during chemotherapy(57.8 compared to 18.2 respectively), a similar increase was seen with5T4. However, unlike 5T4, there was little difference in theproliferative responses detected at weeks 19 and X+2.

1.3 Comparisons of Immunological and Clinical Responses 1.3.1 SummaryImmunological Responses Across all Assays

TABLE 4 Summary immune responses detected in all evaluable patientsTumour Patient Response ID 14 wk X + 8 wk Anecdotal ImmunologicalResponses 101 PR PR ELISA: No 5T4 Abs PROLIF: No response cf screenELISPOT: Very strong to protein, Weak to Pep pool #5 102 CR CR ELISA:Very strong, commencing early and sustained PROLIF: No response cfscreen ELISPOT: Very strong to protein and Class I and II pep 103 PR PRELISA: Moderate, commencing early, increasing after Cx PROLIF: Noresponse cf screen ELISPOT: Weak 104 SD PD ELISA: Weak PROLIF: Moderateand transient post-chemo ELISPOT: Weak 105 PR SD ELISA: Weak, increasingafter Cx PROLIF: Good post-chemo ELISPOT: Very strong to Class I peps107 PR NA ELISA: Very strong, commencing early and sustained PROLIF:Strong post-chemo ELISPOT: Strong and sustained to Class I peps 108 CRCR ELISA: Very strong, commencing early and sustained PROLIF: Moderateand transient ELISPOT: Very strong post chemo to protein and Class I pep113 PD PD ELISA: Weak and transient PROLIF: Strong and sustainedELISPOT: Very Weak 114 CR NA ELISA: Moderate early but not sustainedPROLIF: Strong post-chemo ELISPOT: Weak to protein 116 PR NA ELISA:Moderate Ab response pre, during and post Cx PROLIF: Very strong andsustained throughout ELISPOT: Weak pre and post-Cx 117 SD NA ELISA: WeakPROLIF: Strong and sustained ELISPOT: No response1.3.2 Analysis of Antibody Responses versus Clinical Responses

The MEAN 5T4 antibody titres in all evaluable patients was comparedagainst the reported clinical responses. The mean 5T4 antibody titre wasdetermined over 3 time periods: (i) Over all post TroVax time pointsi.e. from week 2 to week X+10 (or the last data point available), (ii)Over all post-TroVax time points occurring during the period ofchemotherapy (weeks 4-19) and (iii) over all post-TroVax time pointsoccurring following completion of chemotherapy (weeks X+2 to X+10).

Tables 5a-5c: The tables illustrate the mean 5T4 antibody titres overthe entire monitoring period (weeks 2-X+10; Table 5a), duringchemotherapy (weeks 4-19; Table 5b) and following completion ofchemotherapy (weeks X to X+10; Table 5c). The mean titres are rankedfrom LOWEST to HIGHEST alongside the clinical response attributed tothat patient at weeks 14 and X+8.

TABLE 5a Entire time course Mean Post TroVax 5T4 Titres v TumourResopnse Patient Titre 14 wk X + 8 wk 101 0.0 PR PR 104 6.7 SD PD 11315.0 PD PD 105 22.7 PR SD 117 27.5 SD 0 116 31.7 PR 0 114 42.5 CR 0 10366.7 PR PR 102 154.2 CR CR 107 326.7 PR 0 108 395.0 CR CR

TABLE 5b During chemotherapy Mean Post TroVax During Chemo 5T4 Titres vTumour Resopnse Patient Titre 14 wk X + 8 wk 101 0.0 PR PR 105 2.0 PR SD104 3.3 SD PD 117 11.7 SD 0 103 20.0 PR PR 116 21.7 PR 0 113 30.0 PD PD114 80.0 CR 0 102 250.0 CR CR 108 333.3 CR CR 107 360.0 PR 0

TABLE 5c Post chemotherapy Mean Post TroVax Post Chemo 5T4 Titres vTumour Resopnse Patient Titre 14 wk X + 8 wk 101 0 PR PR 113 0 PD PD 1142 CR 0 104 12 SD PD 105 48 PR SD 116 48 PR 0 117 52 SD 0 102 68 CR CR103 136 PR PR 107 352 PR 0 108 548 CR CR

It can be seen that patients with CRs (and many with PRs) fall in thebottom half of the tables i.e. have higher mean 5T4 antibody titres thanthose patients with SD or PD. In table 14c, the post-chemotherapy 5T4titres are reported alongside the tumour responses at week X+8. A numberof X+8wk CT scans are outstanding, but it can be seen that patients withCRs and PRs are clustered at the bottom of the table (i.e. have highpost-chemo Ab titres). The main patient who does not fit with thispattern is patient 101 who did not mount an antibody response orproliferative response to 5T4 but showed a strong ELISPOT response to5T4 protein post chemotherapy and a moderate response to a class Ipeptide.

1.3.3 Analysis of Proliferative Responses Versus Clinical Responses

The MEAN 5T4 proliferative responses (SIs) in all evaluable patientswere compared against the reported clinical responses (some CT scans areoutstanding). The mean proliferative responses were determined over 3time periods (the entire monitoring timecourse, during chemotherapy andfollowing completion of chemotherapy) and are tabulated in tables 6 a-c.

Tables 6a-6c: The tables illustrate the mean 5T4 proliferative responsesover 3 different time periods: Table 6a the entire monitoring period(weeks 2−X+10), Table 6b during chemotherapy (weeks 4-19) and Table 6cfollowing completion of chemotherapy (weeks X to X+10). The meanproliferative responses are ranked from LOWEST to HIGHEST alongside theclinical response attributed to that patient at weeks 14 and X+8.

TABLE 6a Entire time Course Mean Post TroVax 5T4 SIs v Tumour ResopnsePatient SI 14 wk X + 8 wk 104 2.73 SD PD 103 3.52 PR PR 108 4.05 CR CR105 5.16 PR SD 102 5.67 CR CR 101 6.51 PR PR 116 8.34 PR 0 107 8.92 PR 0117 9.64 SD 0 114 9.85 CR 0 113 9.87 PD PD

TABLE 6b During chemotherapy Mean Post TroVax During Chemo 5T4 SIs vTumour Resopnse Patient SI 14 wk X + 8 wk 104 0.79 SD PD 105 1.74 PR SD114 2.34 CR 0 107 2.76 PR 0 103 3.28 PR PR 108 3.63 CR CR 116 4.91 PR 0117 5.31 SD 0 113 5.55 PD PD 102 7.35 CR CR 101 9.20 PR PR

TABLE 6c Post chemotherapy Mean Post TroVax Post Chemo 5T4 SIs v TumourResopnse Patient SI 14 wk X + 8 wk 101 2.18 PR PR 103 4.03 PR PR 1024.73 CR CR 108 5.30 CR CR 104 5.49 SD PD 105 9.98 PR SD 116 14.20 PR 0117 16.43 SD 0 113 16.79 PD PD 107 17.84 PR 0 114 20.75 CR 0

Proliferative responses in this patient group were low duringchemotherapy compared to post-chemotherapy (mean SIs of 4.3 compared to10.4). Furthermore, the greatest mean proliferative response occurred atweek X+10. A potent proliferative response at this time would not impacton tumour responses detected at week X+8, but may have a positive effecton patient survival. Such calculations will be undertaken as the databecome available.

1.3.4 Analysis of ELISPOT Versus Clinical Responses

The MEAN 5T4 ELISPOT responses (antigen specific cells per 10⁶ PBMCs) inall evaluable patients were compared against the reported clinicalresponses. The mean ELISPOT responses were determined at selected timepoints over the entire monitoring time course and are tabulated intables 7 a-c.

Tables 7a-7c: The tables illustrate the mean 5T4 specific ELISPOTresponses (all 5T4 protein and peptide antigens) over the entire timecourse (7a), during chemotherapy (7b) or following completion ofchemotherapy (7c). The mean ELISPOT responses are ranked from LOWEST toHIGHEST alongside the clinical response attributed to that patient atweeks 14 and X+8.

TABLE 7a All 5T4 antigens, entire time course chemo Mean Post TroVax 5T4TOTAL ELISPOT v Tumour Resopnse Patient ELISPOT 14 wk X + 8 wk 113 0 PDPD 117 0 SD 0 103 8.9 PR PR 104 12 SD PD 116 13.8 PR 0 114 42.9 CR 0 10749.3 PR 0 108 159.2 CR CR 105 311.7 PR SD 101 315.2 PR PR 102 431.2 CRCR

TABLE 7b All 5T4 antigens, during Mean Post TroVax 5T4 TOTAL ELISPOTDuring Chemo v Tumour Resopnse Patient ELISPOT 14 wk X + 8 wk 103 0 PRPR 113 0 PD PD 117 0 SD 0 116 7.5 PR 0 104 28.4 SD PD 114 49.4 CR 0 10853 CR CR 107 65 PR 0 101 83.3 PR PR 102 477 CR CR 105 500.8 PR SD

TABLE 7c All 5T4 antigens, post chemo Mean Post TroVax 5T4 TOTAL ELISPOTPost Chemo v Tumour Resopnse Patient ELISPOT 14 wk X + 8 wk 113 0 PD PD117 0 SD 0 104 2.6 SD PD 103 4.4 PR PR 116 20 PR 0 107 42.3 PR 0 11448.8 CR 0 105 247.7 PR SD 101 534 PR PR 108 557.1 CR CR 102 653.3 CR CR

A clustering of positive clinical responses with high mean ELISPOTresponses is observed. In particular, the magnitude of responses to all5T4 antigens over all time points and during the post chemotherapyperiod relates well to the reported tumour responses.

1.3.5 Integrated Analysis of Immunological v Clinical Responses

5T4 specific ELISPOT, proliferation and antibody responses wereintegrated and compared against the reported clinical responses. Toachieve this, each evaluable patient was simply scored relative to themagnitude of the greatest immune response detected. For example, thepatient with the highest mean 5T4 antibody titre across the selectedmonitoring period (i.e. all post TroVax time points, during chemo, postchemo) was given a score of 100%, all other patients were scored as apercentage of the greatest response. To integrate the assays, the scoresper assay for each patient were simply added Such an analysis does takeinto consideration the magnitude of the patients' immune responses butgives equal “weight” to all assays. Tables 8a and b detail the hierarchyof integrated immunological responses (antibody+ELISPOT in table 8a andantibody+ELISPOT+Proliferation in table 8b) versus the reported clinicalresponses.

TABLE 8a Integrated analysis of ELISPOT and Antibody assays over theentire immuno-monitoring time course v clinical responses CombinedTumour Response Titre + Patient 14 wk X + 8 wk ELISPOT Score 113 PD PD3.8 104 SD PD 4.5 117 SD 0 7 116 PR 0 11.2 103 PR PR 19.1 114 CR 0 20.7101 PR PR 73.1 105 PR SD 78.1 107 PR 0 94.1 108 CR CR 136.9 102 CR CR139

TABLE 8b Integrated analysis of ELISPOT, Antibody and Proliferationassays over the entire immuno-monitoring time course v clinicalresponses Tumour Combined Response SI + Titre + Patient 14 wk X + 8 wkELISPOT Score 104 SD PD 32.2 103 PR PR 54.7 116 PR 0 98 113 PD PD 103.8117 SD 0 104.7 114 CR 0 120.5 105 PR SD 130.4 101 PR PR 139 108 CR CR177.9 107 PR 0 184.4 102 CR CR 196.5

1.3.6 Comparisons of Immunological and Clinical Responses: PreliminaryConclusions

This preliminary analysis indicates that some high magnitude 5T4specific immune responses cluster with positive clinical responses. Suchtrends are discussed individually below:

A. ELISA

High 5T4 specific antibody titres are associated more frequently withcomplete and partial responses than with stable or progressive disease.This observation is consistent when responses are analysed over theentire monitoring time course, during and post chemotherapy. Whenanalysing the post-chemo antibody responses, high titres cluster withCRs and PRs at the X+8wk CT scan time point. The main exception to thesetrends is patient 101 who did not sero-convert but who mounted a strongELISPOT response.

B. Proliferation

The magnitude of 5T4 specific proliferative responses does not appear tocluster with positive tumour responses (CRs and PRs). However,proliferative responses were greater following the completion ofchemotherapy, with the greatest overall responses occurring at week X+10i.e. too late to impact on tumour responses. These responses may reflectpositively on survival.

C. ELISPOT

The magnitude of 5T4 specific ELISPOT responses appear to cluster withpositive tumour responses. This trend is evident when the ELISPOT dataare analysed across the entire time course, during chemotherapy andfollowing the completion of chemotherapy.

D. Integrated Analysis: ELISA+ELISPOT

If data from both ELISA and ELISPOT are integrated and the magnitude ofthe combined responses tabulated alongside clinical responses, higherscores cluster more frequently with positive tumour responses. Patientsclassified as SD or PD at 14wk or X+8wk have the lowest combined scores.

E. Integrated Analysis: ELISA+ELISPOT+Proliferation

If data from both ELISA, ELISPOT and proliferation are integrated andthe magnitude of the combined responses tabulated alongside clinicalresponses, again higher scores cluster more frequently with positivetumour responses.

2. TV2 IFL 2.1 Source Clinical and Patient Data

Nineteen patients were recruited into the TV2-FOLFOX trial of whom 12became evaluable for assessment of immunological responses (Table 9).

TABLE 9 Clinical Data.

All 19 ITT patients are included in the table. Evaluable patients areindicated by shading (survival data current to date). Key: WD = Patientwithdrawn therefore no result available; N/A = No data

FIG. 3 shows TV2-IFL: Tumour dimensions throughout the clinical trialtime course. The figure illustrates the sum of the target tumour lesionsat 3 time points: prior to TroVax vaccination (screen) and at weeks Xand X+14.

Of the 11 CT scan datasets available for the 12 evaluable patients, allshowed a decrease in the sum of the target lesions at the Xwk timepoint. By X+14wk, 5 patients (of 7 scans available) had target tumourburdens which remained below the level they were at screening.

2.2 Immunological Responses 2.2.1 Antibody Responses

Table 10 details the 5T4 specific antibody titres for each evaluablepatient across the entire monitoring time course in the TV2-IFL trial.

TABLE 10 5T4 specific antibody responses. Results are expressed as a 5T4specific antibody titre (the greatest serum dilution at which the testsample has a mean O.D. (490 nm) ≧ 2 fold that of the negative controlsample (normal human serum; [NHS]) at each sampling time point. Resultstabulated in bold and ≧10 represent positive antibody responses. Meantitres are reported for all patients at each time point and also atmultiple time points pre-vaccination, during chemotherapy and postchemo. In addition, the fold increase in mean antibody titre compared tothe previous time point is shown after each vaccination. Patient 5T4Specific Antibody Titre at Timepoints (weeks) Post Primary ImmunisationNo −2 0 2 4 6 11 13 17 19 X + 2 X + 4 X + 6 X + 8 X + 10 X + 14 002 <10<10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 003 <10 <10 <10<10 <10 <10 20 20 40 <10 <10 10 20 20 40 005 <10 <10 <10 <10 <10 <10 <10<10 <10 <10 <10 <10 <10 <10 <10 007 <10 <10 <10 160 80 10 10 <10 10 <1020 40 20 10 <10 009 <10 <10 <10 10 <10 <10 <10 <10 <10 <10 10 10 160 80WD 012 <10 <10 10 40 <10 <10 <10 <10 <10 <10 <10 <10 640 640 <10 014 <10<10 <10 20 <10 <10 <10 <10 <10 <10 <10 <10 80 40 40 015 <10 <10 <10 <10<10 <10 <10 <10 <10 <10 <10 <10 80 20 <10 016 <10 <10 <10 10 <10 <10 10<10 <10 <10 40 40 160 80 40 017 <10 <10 <10 <10 <10 <10 <10 <10 <10 <1020 <10 80 80 40 018 <10 <10 <10 <10 <10 <10 <10 <10 40 <10 320 640 51201280 1280 019 <10 <10 10 20 10 <10 10 <10 10 <10 10 10 <10 20 <10 Mean<10 <10 1.7 21.7 7.5 0.8 4.2 1.7 8.3 <10 35 62.5 530 189.2 130.9 TitreFold ↑ 13 5.3 4.9 ? ? 8.5 Mean <10 7.4 158.3 Titre

Over the entire post-TroVax immuno-monitoring time course, the mean 5T4specific antibody titre was observed. The majority of patientssero-converted by week 4 (i.e. following 2 TroVax vaccinations). Themean antibody titres in the In trial were very low during chemotherapy(mean titre of 7.4), decreased following the completion of chemotherapy(between weeks 19 and X+2) and increased dramatically followingcompletion of chemotherapy (mean titre of 158.3). Increases in the meanantibody titre were seen after each of the 6 vaccinations.

2.2.2 Proliferative Responses

Tables 11 a-b detail antigen specific proliferative responses for eachevaluable patient across the entire monitoring time course. Theresponses to 5T4 (11a) and MVA (11b) have been analysed

Tables 11a and b: Summary proliferative responses of PBMCs recoveredfrom patients following in vitro restimulation with 5T4 (a) and MVA (b).Results are expressed as a stimulation index (proliferation induced bymedium alone÷ proliferation induced by test antigen). A stimulationindex ≧2 (indicated by bold text) is considered to be a positiveresponse at that time point. Proliferative responses which are positive(S.I.≧2) and at least 2 fold greater than the pre-injection (−2 or 0week) responses are indicated by red text.

TABLE 11a TV2-IFL: Proliferative responses to 5T4 Sampling Timepoints(weeks) Patient −2 0 2 4 6 11 13 17 19 X + 2 X + 4 X + 6 X + 8 X + 10X + 14 002 1.1 7.1 5.4 3.3 2.7 1 1 1.3 0.44 2.5 2.8 0.8 1.1 1.6 3.11 0036.6 32.8 10.4 17.7 26.7 0.6 0.8 5.7 1.7 1.95 3.6 0.5 11.5 2.62 1.99 0051 4.3 8.9 6.1 1 1.2 0.6 2.7 1.5 0.9 0.4 1.3 1.5 0.6 0.87 007 2.2 6.1 7.61.7 1.3 26.7 4.2 2.1 4.3 1.4 2.7 2.7 0.9 1.52 0.64 009 1.3 17.5 6.1 7.61.3 1.9 1.2 2.1 0.5 1.03 5.21 2.03 0.43 4.50 WD 012 2.2 2.2 2.2 3.1 3.91.5 1.7 2.1 1.55 19.83 22.47 39.70 2.24 14.13 19.64 014 4.2 1.7 17.7 22.6 2 1.2 4.2 2.88 16.58 4.99 5.65 6.04 1.31 10.13 015 2.3 2.8 7 5.6 4.50.5 0.76 1.81 0.73 17.08 3.96 5.02 1.76 2.10 3.94 016 1.9 1.3 7.6 8.60.9 2.7 4.6 1.9 0.92 28.86 3.35 8.41 8.57 3.12 7.73 017 3.5 8 2.1 0.60.9 2.1 3.5 1.27 0.82 6.38 11.30 2.45 4.91 4.71 1.27 018 0.6 3.3 1.3 2.91.3 0.97 0.82 1.14 1.93 12.65 9.65 5.61 5.72 2.80 1.60 019 0.6 2.3 1.581.31 2.72 25.08 10.83 18.67 N/A 0.74 4.01 12.83 1.17 3.00 2.59 Mean 2.37.5 6.5 5 4.1 5.5 2.6 3.7 1.6 9.2 6.2 7.3 3.8 3.5 4.9 SI Fold ↑ 0.9 0.80.5 0.4 5.8 0.7 0.5 Mean 4.9 3.8 5.8 SI

TABLE 11b TV2-IFL: Proliferative responses to MVA Sampling Timepoints(weeks) Patient −2 0 2 4 6 11 13 17 19 X + 2 X + 4 X + 6 X + 8 X + 10X + 14 002 0.4 1.3 3.2 13.7 4.9 1.2 1.7 1.6 1.7 0.4 0.3 5.3 1.1 1 10.41003 1.1 1 10.6 14.8 12 1.8 3.1 0.7 0.4 2.2 29.1 20.4 35.3 52.67 8.60 0052.1 4.5 27 16.2 2 8.7 2.5 1.2 0.4 0.2 0.6 1.5 2.3 4.8 12.50 007 1 1.7 7072.6 6.3 5.8 6.5 1 0.9 1.9 8.6 9.2 42.72 54.25 38.90 009 5.3 21 25.5 2317.8 1.1 0.7 0.7 2.6 44.60 100.56 21.50 83.07 20.31 WD 012 0.8 0.6 1.976.1 5.4 0.7 2.8 1.5 1.63 9.25 43.60 5.28 1.22 45.61 0.93 014 1.1 0.7 110.5 0.8 0.2 1 0.89 15.92 16.49 32.54 24.99 69.54 2.01 7.97 015 0.8 1 11.7 2.3 8.8 4.08 47.44 69.59 14.87 9.44 15.03 7.13 6.23 3.56 016 1.3 12.5 0.6 0.3 1.4 6.3 2.53 15.19 21.58 6.58 12.96 46.31 4.45 11.84 017 0.80.8 1.2 0.1 0.5 0.88 0.30 4.16 4.32 6.13 4.77 1.51 41.83 2.48 1.06 0180.6 2.1 17.7 38.2 14 7.76 3.85 15.01 3.33 3.15 45.89 14.03 25.55 4.7811.18 019 0.2 1 1.49 6.83 53.15 21.13 12.22 12.73 N/A 0.93 4.34 25.101.88 7.97 4.72 Mean 1.3 3.1 14.4 16.2 9.9 5 3.8 7.5 10.5 10.1 23.9 13.129.8 17.2 10.2 SI Fold ↑ 4.6 1.1 0.8 1.4 1 2.4 2.3 Mean 0.95 4 11.9 SI

The mean proliferative responses to 5T4 and MVA show differences in boththe magnitude and longevity of the response. Each antigen will beaddressed in turn:

A. 5T4

Overall, the mean proliferative response to 5T4 showed no increasefollowing TroVax vaccination compared to pre-injection (both have a meanSI of 4.9). Therefore, at the population level and across ALL timepoints, IFL patients show no increase in the mean proliferative responseto 5T4 post-TroVax vaccination. However, at specific time pointsresponses are elevated in individual patients. Responses were elevatedfollowing completion of chemotherapy (S.I. of 5.8 post-chemo versus 3.8during chemo) and this differential was most marked between weeks 19 andX+2 i.e. between the last time point at which most patients were stillreceiving chemotherapy and the first time point following completion ofchemotherapy. This observation was unexpected because patients did notreceive a vaccination within this period, their last vaccination tookplace at week 17. IFL patients showed a 6 fold increase in meanproliferative responses between weeks 19 and X+2 (SI of 1.6 v 9.2respectively) which was the greatest fold increase observed, beinggreater than following any of the 6 vaccinations.

B. MVA

The mean proliferative response to MVA was 6 fold greater followingTroVax vaccination compared to pre-injection (mean S.I. of 13.2 comparedto 2.2 respectively). The MVA specific proliferative response wasgreater following completion of chemotherapy than during chemotherapywhich was similar to the pattern observed for 5T4. However, unlike 5T4,there was no difference in the proliferative response detected at weeks19 and X+2 (mean S.I.s of 10.5 versus 10.1 respectively).

2.3 Comparisons of Immunological and Clinical Responses 2.3.1 SummaryImmunological Responses Across all Assays

TABLE 12 Summary immune responses seen in all evaluable patients TumourPatient Response ID Xwk X + 14 wk Summary Immunological Responses 002 CRCR ELISA: No 5T4 Ab response detected PROLIF: No response cf screenELISPOT: Weak response single time point to peptide #77 003 PR PD ELISA:Weak Ab response during and post-chemotherapy PROLIF: No response cfscreen ELISPOT: Very weak to 5T4 and pool #8 at X + 8 wk only 005 PR PDELISA: No 5T4 Ab response detected PROLIF: Weak and transient responsecf screen ELISPOT: Strong responses pre and post Cx to multiple Peps 007SD PD ELISA: Weak Ab response pre Cx, moderate post Cx PROLIF: Weak buttransient response cf screen ELISPOT: Very strong to 5T4 protein only.Post Cx 009 SD PD ELISA: Weak Ab response pre Cx, moderate post CxPROLIF: No response cf screen ELISPOT: Very strong to Class I peps preand post Cx 012 PR PD ELISA: Weak Ab response pre Cx, strong post CxPROLIF: Strong response but only post-chemo ELISPOT: Very strong tomultiple peps pre and post Cx 014 SD ELISA: Weak Ab response pre Cx,moderate post Cx PROLIF: Strong response pre and post-chemo ELISPOT: Noresponses 015 PR PD ELISA: No Ab response pre or during Cx, moderatepost Cx PROLIF: Strong but transient response pre and post-chemoELISPOT: Very Weak to one class II peptide at one timepoint  16 PR PDELISA: Weak Ab response pre Cx, moderate post Cx PROLIF: Strong responsepre, during but primarily post-Cx ELISPOT: Weak response to class IIpeptide #41 017 PR PD ELISA: No Ab response pre or during Cx, moderatepost Cx PROLIF: No response cf screen ELISPOT: Very weak to peptide pool#13 @ 1 timepoint 018 SD PD ELISA: No Ab response pre Cx, very strongpost Cx PROLIF: Strong transient response post-Cx ELISPOT: Very strongand sustained class I pools 1, 5 and 20 019 PD PD ELISA: Very weak Abresponse throughout PROLIF: Strong response during and post-Cx ELISPOT:Very weak single timepoint

2.3.2 Analysis of Antibody Responses Versus Clinical Responses

The MEAN 5T4 antibody titres in all evaluable patients were comparedagainst the reported clinical responses (some CT scans are outstanding).Mean 5T4 antibody titres were determined over 3 time periods: (i) Overall post TroVax time points i.e. from week 2 to week X+10 (or the lastdata point available), (ii) Over all post-TroVax time points occurringduring the period of chemotherapy (weeks 4-19) and (iii) over allpost-TroVax time points occurring following completion of chemotherapy(weeks X+2 to X+14). Tables 13a-13c rank the mean antibody titresagainst the respective clinical responses reported in that patient.

Tables 13a-13c: The tables illustrate the mean 5T4 antibody titres overthe entire monitoring period (weeks 2-X+14; Table 13a), duringchemotherapy (weeks 4-19; Table 13b) and following completion ofchemotherapy (weeks X to X+14; Table 13c). The mean titres are rankedfrom LOWEST to HIGHEST alongside the clinical response attributed tothat patient at weeks X and X+14.

TABLE 13a Entire Time Course Mean Post TroVax 5T4 Titres v TumourResponse Patient Titre Xwk X + 14 wk 2 0.0 CR CR 5 0.0 PR PD 15 7.7 PRPD 19 7.7 PD PD 3 13.1 PR PD 14 13.8 SD 17 16.9 PR PD 9 22.5 SD PD 727.7 SD PD 16 29.2 PR PD 12 102.3 PR PD 18 667.7 SD PD

TABLE 13b During Chemotherapy Mean Post TroVax 5T4 Titres During Chemo vTumour Response Patient Titre Xwk X + 14 wk 2 0.0 CR CR 5 0.0 PR PD 150.0 PR PD 17 0.0 PR PD 9 1.7 SD PD 14 3.3 SD 16 3.3 PR PD 12 6.7 PR PD18 6.7 SD PD 19 8.3 PD PD 3 13.3 PR PD 7 45.0 SD PD

TABLE 13c Post Chemotherapy Mean Post TroVax 5T4 Titres Post Chemo vTumour Response Patient Titre Xwk X + 14 wk 2 0.0 CR CR 5 0.0 PR PD 196.7 PD PD 3 15.0 PR PD 7 15.0 SD PD 15 16.7 PR PD 14 26.7 SD 17 36.7 PRPD 9 52.0 SD PD 16 60.0 PR PD 12 213.3 PR PD 18 1440.0 SD PD

Antibody responses in this patient group were very low duringchemotherapy compared to post-chemotherapy (mean titres of 7 compared to158). Furthermore, the greatest mean antibody titre occurred at weekX+8. A potent antibody response at this time may not impact on tumourresponses detected at week X+14, but may have a positive effect onpatient survival.

2.3.3 Analysis of Proliferative Responses Versus Clinical Responses

The MEAN 5T4 proliferative responses (SIs) in all evaluable patientswere compared against the reported clinical responses (some CT scans areoutstanding). The mean proliferative responses were determined over 3time periods (the entire monitoring timecourse, during chemotherapy andfollowing completion of chemotherapy) and are tabulated in tables 14a-c.

Tables 14a-14c: The tables illustrate the mean 5T4 proliferativeresponses over 3 different time periods: Table 14a the entire monitoringperiod (weeks 2-X+14), Table 14b during chemotherapy (weeks 4-19) andTable 14c following completion of chemotherapy (weeks X to X+14). Themean proliferative responses are ranked from LOWEST to HIGHEST alongsidethe clinical response attributed to that patient at weeks X and X+14.

TABLE 14a Entire Time course Mean Post TroVax 5T4 SI v Tumour ResopnsePateient SI Xwk X + 14 wk 2 2.08 CR CR 5 2.12 PR PD 9 2.83 SD PD 17 3.25PR PD 18 3.72 SD PD 15 4.21 PR PD 7 4.44 SD PD 14 5.94 SD 3 6.60 PR PD16 6.71 PR PD 19 7.04 PD PD 12 10.31 PR PD

TABLE 14b During Chemotherapy Mean Post TroVax 5T4 SI During Chemo vTumour Resopnse Pateient SI Xwk X + 14 wk 18 1.51 SD PD 17 1.53 PR PD 21.62 CR CR 5 2.18 PR PD 12 2.31 PR PD 15 2.32 PR PD 9 2.43 SD PD 14 2.48SD 16 3.27 PR PD 7 6.72 SD PD 3 8.87 PR PD 19 11.72 PD PD

TABLE 14c Post Chemotherapy Mean Post TroVax 5T4 SI Post Chemo v TumourResopnse Pateient SI Xwk X + 14 wk 5 0.93 PR PD 7 1.64 SD PD 2 1.99 CRCR 9 2.64 SD PD 3 3.69 PR PD 19 4.06 PD PD 17 5.17 PR PD 15 5.64 PR PD18 6.34 SD PD 14 7.45 SD 16 10.01 PR PD 12 19.67 PR PD

2.3.4 Analysis of Elispot Responses Versus Clinical Responses

The MEAN 5T4 ELISPOT responses (antigen specific cells per 10⁶ PBMCs) inall evaluable patients were compared against the reported clinicalresponses (some CT scans are outstanding). The mean ELISPOT responseswere determined at selected time points over the entire monitoring timecourse and are tabulated in tables 15 a-c.

Tables 15a-15c: The tables illustrate the mean 5T4 ELISPOT responsesover the entire time course (15a), during chemotherapy (15b) orpost-chemotherapy (15c). The mean ELISPOT responses are ranked fromLOWEST to HIGHEST alongside the clinical response attributed to thatpatient at weeks X and X+14.

TABLE 15a All 5T4 antigens, entire time course Mean Post TroVax 5T4TOTAL ELISPOT v Tumour Response Patient ELISPOT Xwk X + 14 wk 2 47.5 CRCR 3 6.7 PR PD 5 91.6 PR PD 7 33.5 SD PD 9 371.8 SD PD 12 99.2 PR PD 140 SD 15 7.2 PR PD 16 33.5 PR PD 17 5.7 PR PD 18 165.6 SD PD 19 4.1 PD PD

TABLE 15b All 5T4 antigens, during chemotherapy Mean Post TroVax DURINGChemo 5T4 ELISPOT v Tumour Response Patient ELISPOT Xwk X + 14 wk 3 0 PRPD 7 0 SD PD 16 0 PR PD 17 0 PR PD 14 0 SD 19 13.6 PD PD 15 14.4 PR PD12 26.3 PR PD 18 79.7 SD PD 5 91.7 PR PD 2 113.25 CR CR 9 264.4 SD PD

TABLE 15c All 5T4 antigens, post chemotherapy Mean Post TroVax POSTChemo 5T4 ELISPOT v Tumour Resopnse Patient ELISPOT Xwk X + 14 wk 15 0PR PD 19 0 PD PD 14 0 SD 3 8.9 PR PD 17 13.3 PR PD 2 26.5 CR CR 7 50.3SD PD 16 55.8 PR PD 5 91.6 PR PD 12 189.3 PR PD 18 236.1 SD PD 9 425.4SD PD

Analysis of 5T4 ELISPOT responses showed a better clustering of high 5T4responses with beneficial clinical responses than did the samecalculation using proliferation as a variable.

2.3.5 Integrated Analysis of Immunological v Clinical Responses

5T4 specific ELISPOT, proliferation and antibody responses wereintegrated and compared against the reported clinical responses. Toachieve this, each evaluable patient was simply scored relative to themagnitude of the greatest immune response detected. For example, thepatient with the highest mean 5T4 antibody titre across the selectedmonitoring period (i.e. all post TroVax time points, during chemo, postchemo) was given a score of 100%, all other patients were scored as apercentage of the greatest response. To integrate the assays, the scoresper assay for each patient were simply added Such an analysis does takeinto consideration the magnitude of the patients' immune responses butgives equal “weight” to all assays. Tables 16 a and b detail thehierarchy of integrated immunological responses (antibody+ELISPOT intable 16a and antibody+ELISPOT+Proliferation in table 16b) versus thereported clinical responses.

TABLE 16a Integrated analysis of ELISPOT + Antibody assays over theentire immuno-monitoring time course v clinical responses TumourCombined Response Titre + ELISPOT Patient Xwk X + 14 wk Score 14 SD 2 19PD PD 2 15 PR PD 3 3 PR PD 4 17 PR PD 5 2 CR CR 13 7 SD PD 13 16 PR PD13 5 PR PD 25 12 PR PD 42 9 SD PD 103 18 SD PD 145

TABLE 16b Integrated analysis of ELISPOT, Antibody and Proliferationassays over the entire immuno-monitoring time course v clinicalresponses Tumour Combined Response SI + Titre + ELISPOT Patient Xwk X +14 wk Score 2 CR CR 33 17 PR PD 37 15 PR PD 44 5 PR PD 46 7 SD PD 56 14SD 60 3 PR PD 68 19 PD PD 70 16 PR PD 78 9 SD PD 130 12 PR PD 142 18 SDPD 181

Strong immune responses in the IFL trial occurred primarily followingcompletion of chemotherapy. Therefore it is unlikely that the immuneresponse would have a beneficial impact on the tumour by the X week CTscan and possibly not even by X+14.

3. A Comparison of Immune Responses Occurring in the FOLFOX and IFLTrials 3.1 Antibody Responses

Over the entire immuno-monitoring time course, the mean 5T4 specificantibody titres (to date) were similar for both trials, 76 for TV2-IFLand 100 for TV2-FOLFOX (Table 17). However, the kinetics of the antibodyresponses were different for each trial. In the IFL trial, antibodyresponses were very low during chemotherapy (a mean titre of 7 betweenweeks 4 to 19) but increased dramatically (>20 fold) followingcompletion of chemotherapy (a mean titre of 158 between weeks X+2 andX+14). In contrast, mean antibody titres in the FOLFOX trial were highduring chemotherapy (mean titre of 103) and increased moderatelyfollowing completion of chemotherapy (mean titre of 115). In bothtrials, the mean antibody titres decreased between weeks 19 and X+2.Increases in antibody titre were seen after all 6 vaccinations in bothtrials.

TABLE 17 A comparison of 5T4 specific antibody responses between TV2 IFLand FOLFOX trials. Mean Response Measurement of 5T4 Specific FOLFOX IFLAntibody Responses (n = 11) (n = 12) Mean 5T4 Ab response (pre TroVax)<10 <10 Mean 5T4 Ab response (all time points) 100 76.1 Mean 5T4 Abresponse (during chemo) 102.6 7.4 Mean 5T4 Ab response (post chemo)115.1 158.3

3.1.1 Statistical Analysis of 5T4 Specific Antibody Responses Detectedin TV2 IFL and FOLFOX Trials

A statistical analysis of the data was undertaken on a “per trial” basisusing Wilcoxon. Results are as follows:

(a) Response During Chemotherapy (Weeks 2 to 19)

IFL: mean score (titre)=0.44 (<10)FOLFOX: mean score (titre)=1.73 (17)Difference between groups P=0.011 (P<2%)Conclusion: There is a significant difference in the antibody titresdetected in the IFL and FOLFOX groups during the period in whichpatients receive chemotherapy.(b) Response after Chemotherapy (Weeks X+2 to X+10)IFL: mean score (titre)=1.85 (18)FOLFOX: mean score (titre)=2.29 (24)Difference between groups P=0.53 (NS)Conclusion: There is no significant difference in the antibody titresdetected in the IFL and FOLFOX groups during the period followingcompletion of chemotherapy.(c) Change from During to after Chemotherapy (Weeks 2-19 v X+2-X+10)IFL: mean score (titre) during=0.44 (<10) after=1.85 (18) change=1.41(×2.7) P=0.020 (P<2%)FOLFOX: mean score (titre) during 1.73 (17) after=2.29(24)change=0.56(×1.5) P=0.16 (NS)Conclusion: There is a significant difference in the antibody titresdetected in the IFL group during chemotherapy compared topost-chemotherapy. In contrast, there is no significant difference inthe FOLFOX group.(d) Change from Week 19 to Week X+2

IFL:

mean score (titre) week 19=0.67 (<10)mean score (titre) week X+2=0.00 (<10)change=−0.67 (×0.62) P=0.13 (NS)

FOLFOX:

mean score (titre) week 19=2.27 (24)mean score (titre) week X+2=0.36 (<10)change=−1.91 (4.27) P=0.016 (P<2%)difference between groups P=0.10 (NS)Conclusion: Between weeks 19 and X+2 there is no significant differencein the antibody titres detected in the IFL group but a significantdecrease in the titres in the FOLFOX group.

3.2 Proliferative Responses

5T4 specific proliferative responses were of greater magnitude in theFOLFOX compared to the IFL trial (Table 18). In both trials,proliferative responses were of greater magnitude following completionof chemotherapy. The differential was most profound between weeks 19 andX+2 as illustrated in FIGS. 4 a-b. However, such differences were not asapparent when the responses to MVA or TT were analysed (FIGS. 4 c-4 f).

TABLE 18 A comparison of 5T4 specific proliferative responses betweenTV2 IFL and FOLFOX trials. Mean Response Measurement of 5T4 SpecificFOLFOX IFL Immune Response (n = 11) (n = 12) Mean 5T4 proliferativeresponse (pre TroVax) 3.6 4.9 Mean 5T4 proliferative response (all timepoints) 6.8 4.9 Mean 5T4 proliferative response (during chemo) 4.3 3.8Mean 5T4 proliferative response (post chemo) 10.5 5.8

FIG. 4 a shows 5T4 Responses in TV2-IFL, FIG. 4 b shows 5T4 Responses inTV2-FOLFOX, FIG. 4 c shows MVA Responses in TV2-IFL, FIG. 4 d shows MVAResponses in TV2-FOLFOX, FIG. 4 e shows TT Responses in TV2-IFL and FIG.4 f shows TT Responses in TV2-FOLFOX

3.2.1 Statistical Analysis of 5T4 Specific Proliferative ResponsesDetected in TV2 IFL and FOLFOX Trials

A statistical analysis (Wilcoxon) was undertaken to assess thesignificance of antigen-specific immune responses detected in TV2 FQLFOXand IFL trials.

-   (a) change in proliferative responses from baseline to    post-vaccination (weeks −2 and 0 versus weeks 2 to week X+14 (IFL)    or week X+10 (FOLFOX))    5T4 IFL: mean log(S.I.) baseline=1.05 (2.9) post-v=1.02 (2.8)    change=−0.03 (×0.97) P=0.96 (NS)    FQLFOX: mean log(S.I.) baseline=0.69 (2.0) post-v=1.25 (3.5)    change=0.56 (×1.8) P=0.10 (NS)

MVA

IFL: mean log(S.I.) baseline=0.17 (1.2) post-v=1.62 (5.1) change=1.45(×4.3) P=0.002 (P<0.5%)FOLFOX: mean log(S.I.) baseline=0.18 (1.2) post-v=2.37 (10) change=2.19(×8.9) P=0.002 (P<0.5%)

TT

IFL: mean log(S.I.) baseline=0.90 (2.5) post-v=1.06 (2.9) change=0.17(×1.2) P=0.52 (NS)FOLFOX: mean log(S.I.) baseline=1.42 (4.1) post-v=1.50 (4.5) change=0.08(×1.1) P=1.00 (NS)Conclusion: Proliferative responses to 5T4 and TT show no significantchange from baseline to post-TroVax vaccination. However, responses toMVA show a significant increase.

-   (b) change in proliferative responses from baseline to during    chemotherapy    (weeks −2 and 0 versus weeks 4 to week 19)

5T4

IFL: mean log(S.I.) baseline=1.05 (2.9) during=0.76 (2.2) change=−0.29(×0.75) P=0.11 (NS)FOLFOX: mean log(S.I.) baseline=0.69 (2.0) during=0.94 (2.6) change=0.25(×1.3) P=0.64 (NS)

MVA

IFL: mean log(S.I.) baseline=0.17 (1.2) during=1.17 (3.2) change=1.00(×2.7) P.03 (P<5%)FOLFOX: mean log(S.I.) baseline=0.18 (1.2) during=1.99 (7.3) change=1.81(×6.1) P=0.003 (P<0.5%)

TT

IFL: mean log(S.I.) baseline=0.90 (2.5) during=0.98 (2.7) change=0.09(×1.1) P=0.79 (NS)FOLFOX: mean log(S.I.) baseline=1.42 (4.1) during=1.47 (4.3) change=0.04(×1.04) P=0.89 (NS)Conclusion: Proliferative responses to 5T4 and TT show no significantchange from baseline to post-TroVax vaccination time points occurringduring chemotherapy. However, responses to MVA show a significantincrease.

-   (c) change in proliferative responses from baseline to after    chemotherapy    (weeks −2 and 0 versus weeks 2 to week X+14 (IFL) or week X+10    (FOLFOX))

5T4

IFL: mean log(S.I.) baseline=1.05 (2.9) after =1.18 (3.3) change=0.13(×1.1) P1.79 (NS)FOLFOX: mean log(S.I.) baseline=0.69 (2.0) after =1.82 (6.2) change=1.13(×3.1) P=0.04 (P<5%)

MVA

IFL: mean log(S.I.) baseline=0.17 (1.2) after =2.05 (7.8) change=1.88(×6.6) P=0.001 (P<0.2%)FOLFOX: mean log(S.I.) baseline=0.18 (1.2) after =2.84 (17) change=2.66(×14) P=0.003 (P<0.5%)

TT

IFL: mean log(S.I.) baseline=0.90 (2.5) after =1.16 (3.2) change=0.27(×1.3) P=0.27 (NS)FOLFOX: mean log(S.I.) baseline=1.42 (4.1) after =1.58 (4.9) change=0.16(×1.2) P=0.90 (NS)Conclusion: Proliferative responses to 5T4 (IFL only) and TT show nosignificant change from baseline to post-TroVax vaccination time pointsoccurring following completion of chemotherapy. However, responses toMVA and 5T4 (FOLFOX only) show a significant increase.

-   (d) change in proliferative responses between weeks 19 and X+2

5T4

IFL: mean log(S.I.) week 19=0.33 (1.4) week X+2=1.49 (4.4) change=1.16(×3.2) P=0.04 (P<5%)FOLFOX: mean log(S.I.) week 19=0.75 (2.1) week X+2=1.64 (5.2)change=0.89 (×2.4) P41.04 (P<5%)

MVA

IFL: mean log(S.I.) week 19=1.24 (3.5) week X+2=1.37 (3.9) change=0.13(×1.1) P=0.68 (NS)FOLFOX: mean log(S.I.) week 19=2.02 (7.5) week X+2=2.33 (10) change=0.31(×1.4) P=0.64 (NS)

TT

IFL: mean log(S.I.) week 19=1.42 (4.1) week X+2=1.13 (3.1) change=−0.29(×0.75) P=0.34 (NS)

FOLFOX: mean log(S.I.) week 19=1.48 (4.4) week X+2=1.47 (4.3)change=−0.02 (×0.98) P=0.90 (NS)

Conclusion: Proliferative responses to MVA and TT show no significantchange from week 19 to X+2. However, responses to 5T4 show a significantincrease.

Summary

Antibody responses in the IFL group are suppressed by the chemo regimencompared to the FOLFOX group. However, in both groups a boosting effectcan be seen in many patients following the 5^(th) and 6^(th)vaccinations—this was rarely seen in the previous TV1 trial. Indeed,mean 5T4 antibody titres were boosted following each of the 6vaccinations in both IFL and FOLFOX trials.

At the cellular level, mean proliferative responses to both 5T4 and TTshowed no significant increase post-TroVax vaccination compared tobaseline levels. However, mean proliferaitve responses detectedfollowing completion of chemotherapy were significantly elevatedcompared to baseline in the FOLFOX trial. The increase in proliferativeresponses to 5T4 between weeks 19 and X+2 is intriguing. Responses to5T4, but not MVA or TT, were significantly enhanced between these 2 timepoints despite the fact that no vaccinations occurred within this period(approximately 8 weeks). An analysis of the levels of regulatory T cellsin the periphery may provide further insight into this observation.

4. T Regulatory Cells

A subset of T cells was originally referred to as suppressor T cells inthe 1970s due to their ability to induce tolerance and are now describedas T regulatory cells (Gershon and Kondo, 1970. Immunology 18:723-737;Gershon 1975. Transplant Rev. 26:170-185; Taams and Akbar, 2005. Curr.Top. Microbiol. Immunol. 293:115-131). T regulatory (Tr) cells play anessential role in the induction and maintenance of tolerance to bothforeign and self antigens. Different types of regulatory/suppressorcells have been described, including CD4⁺CD25⁺ T cells, TGF-β producingTH3 cells, IL-10 producing Tr1 cells and CD8⁺CD28⁻ T cells (for reviewssee Levings and Roncarlo, 2005. CTMI. 292:303-326; Huehn, Siegmund andHamann, 2005. CITR 293: 89-114; Faria and Weiner, 2005 Immunol. Rev.206:232-259; Weiner, 2001 Immunol. Rev. 182:207-214; Weiner et al, 2001.Microbes Infect. 3: 947-954; Roncarolo et al, 2001 Immunol. Rev. 182:68-79

4.1. CD4⁺CD25⁺ Tregs: Introduction

These regulatory T cells can be detected in human peripheral blood andare able to suppress bystander T cells in an antigen non-specific andcontact-dependent manner. These Tregs are characterised by expression ahigh constitutive levels of the α-chain (CD25) of the IL-2 receptor andare often referred to as CD4⁺CD25^(hi+) Tregs. They also express highlevels of the intracellular transcription factor FoxP3 and other cellreceptors including CTLA-4, GITR (Glucocorticoid-induced TGFRsuperfamily member 18), CD45RO, CD45R13, ICOS and neuropilin 1. Incontrast to murine CD4⁺CD25⁺ Tregs, human counterparts do not expresshigh levels of the integrin CD103. Since all of the markers that can beused to identify CD4⁺CD25⁺cells can be expressed by other subsets of Tcells, albeit in various levels and combinations, there is no singlemarker that defines a CD4⁺CD25⁺ Treg cell.

The precise molecular mechanism(s) by which CD4⁺CD25⁺ Tregs exert theirsuppressive function remains undefined, but requires cell contact and isnot dependent on the Tregs secreting cytokines themselves (Takahashi etal, 1998 Int. Immunol. 10:1969-1980; Thorton and Shevach 1998. J. Exp.Med. 188:287-296). CD4⁺CD25⁺ Tregs can exert their suppressive effectson T cells, B cells, dendritic cells, NK cells (Shimuzu et al, 1999. J.Immunol. 163: 5211-5218), neutrophils, monocytes and macrophages (Maloyet al, 2003. J. Exp. Med. 197: 111-119; Taams et al, 2005. Hum. hnmunol.66: 222-230).

CD4⁺CD25⁺ Tregs may inhibit the induction and effector activities ofboth CD4⁺and CD8⁺(For review see Van Boehmer 2005. Nat. Immunol.6:338-344). The modes of actions of these Tregs appear to vary and mayinclude control of cytokine secretion (e.g. IL-2 and IFN-γ) from theeffector T cells, suppression of cyolytic killing by CD8⁺ T cells (Chenet al, PNAS 102:419-424), interference with receptor signalling, killingof effector T cells by a perforin dependent mechanism (Piccirillo andShevach, 2004. Semin. Immunol. 16:81-88) and induction of IL-10 andTGF-β secreting T cells Jonuleit et al, 2002 J. Exp. Med. 196:255-260).Some of the later cells appear to be Tr1 Tregs (Levings et al, 2002 Int.Arch. Allergy Immunol. 129: 262-276) suggesting that CD4⁺CD25⁺ Tregspromote differentiation of other Tregs.

B cell activities can also be regulated by CD4⁺CD25⁺ Tregs. These Tregshave been found to suppress the maturation of autoantibody responses(Fields et al, 2005 J. Immunol. 175:4255-4264), activation and antibodysecretion of B cells (Sakaguchi et al, 1995. J. Immunol. 155:1151-1164;Bystry et al, 2001. Nat. Immunol. 2:1126-1132). Killing of B cellsinvolved in antigen presentation by Tregs via Fas/FasL interactions hasalso been reported (Janssens et al, 2003 J. Immununol. 171:4604-4612).

CD4⁺CD25⁺ Tregs may administer their suppressive effects on bystandercells by regulating the actions of antigen presenting cells. CD4⁺CD25⁺Tregs may limit the stimulatory capacity of APCs by down-regulating cellsurface expression of costimulatory molecules such as CD80 and CD86and/or preventing maturity (Cederborn et al, 2000. Eur. J. Immunol.30:1538-1543; Grundstorm et al, 2003. J. Immunol. 170:5008-5017; Taamset al, 2005. Hum. Immunol. 66:222-230; Misra et al, 2004. J. Immunol.172:4676-4680). In another study, Tregs suppressed myeloid DCmaturation, by both blocking costimulatory molecule up-regulation andinhibiting cytokine secretion, which resulted in poor antigenpresentation capacity (Hout et al, 2006. J. Immunol. 176:5293-5298).However, plasmacytoid DCs, that favour TH2 development, were insensitiveto the actions of Tregs. Poor antigen presentation by monocytes (Taamset al, 2005. Hum. Immunol. 66:222-230) and monocyte-derived DC driven inthe presence of Tregs has also been described (Misra et al, 2004. J.Immunol. 172:4676-4680). Tregs may also exert a negative feedbackmechanism on Th1-type responses induced by mature DCs, thereby dampeningthe development of TH1 responses (Oldenhove et al, 2003. J. Exp. Med.198:259-266). In addition, Tregs may regulate a DCs ability to secreteindoleamine 2,3-dioxygenase (IDO), an enzyme that catabolises thedepletion of the essential amino acid tryptophan and enhances theproduction of kynurenine that inhibit T cell proliferation and promotepreferential apoptosis of activated T cells (Terness et al, 2002. J.Exp. Med. 196:447-457). High levels of IDO results in depriving T cellsof tryptophan and subsequent apoptosis (Fallarino et al, 2003. Nat.Immunol. 4:1206-1212). It has also been shown that the Tregs restrictcontact between DCs and CD4+helper cells (Tand and Krummel, 2006. Curr.Opin. Immun. 18: 496-502).

Evidence suggests that CD4⁺CD25⁺ Tregs arise from the thymus and maydifferentiate from CD4⁺CD25⁻ T cells in the periphery (For review seeHuehn, Siegmun and Hamann, 2005. CTMI 293:89-114; Taams and Akbar, 2005CTMI 293: 115-131; Akbar et al, 2003 Immunology 109:319-325; Bluestoneand Abbas, 2003). FoxP3 expression is critical for the development andfunction of CD4⁺CD25⁺ Tregs (for review see Nomura and Sakaguchi, 2005.CTMI 293:287-302). In addition, numerous studies demonstrate that avariety of interactions between CD4⁺CD25⁺ Tregs and APCs are requiredfor the development and function of Tregs (Rutella and Lemoli, 2004.Immunol. Lett. 94:11-26; Zheng et al, 2004 J.I. 173:2428; Herman et al,2004. J. Exp. Med. 199:1479; Min et al, 2003. J. Immunol. 170:1304-1312;Kumanogoh et al, 2001. J.I. 166:353; Salomom et al, 2000). For example,Tregs fail to develop in mice that lack CD28/B7 interactions. However,CD4⁺CD25⁺ Tregs may also affect the differentiation of DC (Min et al,2003. J. Immunol. 170:1304-1312).

The antigen repertoire of CD4⁺CD25⁺ Tregs is thought to be as broad asthat of naïve T cells, enabling recognition of a wide array of bothself- and non-self-antigens, thus allowing control of various immuneresponses. In order to exert their suppressive capacity, activation oftheir TCR is required. However, once activated they can suppress antigennon-specifically (Jonuleit et al, 1999. J. Immunol. 193:1285; Thorntonand Shevach, 2000. J. Inununol. 164: 183-190; Levings, Sangregorio andRoncarolo, 2001 J. Exp. Med. 193:1295; Yamagiwa et al, 2001 J. Immunol.166:7282). After TCR-mediated stimulation, the same Tregs suppressed theactivation of naïve CD4⁺CD25− T cells activated by alloantigens andmitogens (Levings, 2001) Evidence also suggests that peripheral antigenis required for the development, maintenance, or expansion of some Tregs(Mastellar, Tang and Bluestone 2006. Semin. Immunol. 18:103-110).

4.2. Analysis of CD4⁺CD25⁺ Tregs in PBMC from TV2 Patients

Levels of CD4⁺CD25⁺ Tregs were estimated by staining PBMCs with anti-CD4and -CD25 antibodies, gating on CD4⁺cells and setting a strict quadrantin order to only include CD4⁺CD25^(hi+) cells as a positive readout, Thesame quadrant was used to evaluate all patients. An example of stainingis shown in FIG. 5.

Intracellular staining was also performed to confirm that these cellsexpressed the transcription factor FoxP3 (FIG. 6).

In the literature, it is generally accepted that levels of CD4⁺CD25⁺Tregs are represented as a percentage of the total CD4⁺ T cellpopulation. Although there is some debate, CD4⁺CD25⁺ Treg cellsrepresent up to a 2 to 10% maximum of CD4⁺ T cells present in peripheralblood of healthy individuals (Maloy and Powrie 2001. Nat. Immunol.2:816-822; Sakaguchi et al, 2001. Immunl. Rev. 182: 18-32; Gavin andRudensky, 2003. Curr. Opin. Immunol. 15:690-696; Piccirillo and Shevach,2004.

Seim. Immunol. 16:81-88 Masteller, Tang and Bluestone 2006. Semin.Immunol. 18: 103-110).

Data for TV2-IFL and -FOLFOX patients are tabulated in Table 1. Patientsappeared to comprise normal levels of CD4⁺CD25⁺ Tregs (up to 2-10%)within their peripheral blood CD4⁺ T cells prior to chemotherapy.

TABLE 19 Percentage of CD4⁺CD25⁺ Tregs in the total CD4⁺ T cellpopulation. Means for stage of trial are calculated as the average ofeach individual time point. Time Point (weeks) Patient No. −2 0 2 4 6 1113 17 19 X + 2 X + 4 X + 6 X + 8 X + 10 X + 14 TV2-002 0.99 1.22 0.660.60 0.77 0.87 TV2-003 1.20 0.64 0.67 0.54 0.43 2.29 TV2-012 0.67 0.530.29 0.27 0.34 0.99 TV2-014 2.10 2.15 0.98 1.84 2.27 TV2-016 1.34 2.410.59 1.06 0.95 1.61 TV2-018 0.87 1.27 0.55 0.14 0.37 0.63 TV2-019 3.683.80 3.06 2.90 1.98 3.09 IFL Mean 1.01 2.89 0.64 1.90 0.55 3.06 0.600.98 0.95 0.99 1.79 IFL Mean During 1.55 1.72 0.95 1.32 Trial StageTV2-101 2.63 1.90 1.25 1.62 1.73 2.85 TV2-104 1.43 0.81 0.59 1.02 0.750.85 TV2-107 2.44 1.85 2.26 2.75 2.41 3.33 TV2-108 2.38 0.86 1.59 1.372.44 1.46 TV2-113 3.27 2.54 1.31 2.59 2.17 2.33 TV2-117 4.40 3.01 3.624.04 5.02 5.69 FOLFOX Mean 1.43 2.78 4.40 1.73 2.14 1.48 1.64 2.59 2.082.26 4.18 1.87 5.69 FOLFOX Mean 2.44 2.40 1.66 2.70 During Trial StagePre-Trial Pre-chemo Chemotherapy Post-Chemotherapy

Both IFL and FOLFOX Treatment Resulted in Depletion of Tregs.

For the IFL patients (n=7) Tregs decreased during chemotherapy in therange of 15-83% compared to pre-chemotherapy time points. For the FOLFOXpatients (n=5), the range observed was 31-52% (note patient TV2-108excluded as there was no pre-chemotherapy time point examined).

Following chemotherapy, levels of Tregs increased compared to thechemotherapy stage, suggesting that the effects of both IFL and FOLFOXare transient. In some patients, levels of Tregs returned to pre-triallevels.

The means of the percentage of CD4⁺CD25⁺ Tregs in the CD4⁺populationsduring various stages of the trial has been plotted in FIG. 7. Ingeneral, the group of TV2-FOLFOX patients displayed higher levels ofCD4⁺CD25⁺ Tregs than the group of TV2-IFL patients investigated.Although the levels of Tregs were higher in the TV2-FOLFOX patients (butwithin the expected range for healthy individuals), than the TV2-IFLpatients, the mean changes that were observed throughout the trial inthis cell population are similar for both trials. Tregs decreased duringchemotherapy by approximately 30% compared to the pre-trial stage.Following chemotherapy, levels of these cells increased but remainwithin the expected normal range of CD4⁺CD25⁺ Tregs.

Statistical analysis of the data revealed that the percentage ofCD4⁺CD25⁺ Tregs in the CD4⁺ T cell population decreased significantlyduring chemotherapy compared to pre-chemotherapy in both trials. Thegeometric mean of Tregs decreased from 1.36 before chemotherapy to 0.68during IFL treatment (p=0.001). The geometric mean of Tregs decreasedfrom 2.31 prior to chemotherapy to 1.75 during FOLFOX treatment(p=0.0093).

However, levels of Tregs following chemotherapy were not significantlydifferent to that before chemotherapy. The geometric mean of Tregspost-chemotherapy was 1.06 (p=0.2629) and 2.17 (p=0.4923) for the IFLand FOLFOX trials, respectively. This data implies that followingdepletion of CD4⁺CD25⁺ Tregs during either IFL and FOLFOX treatment,levels of Tregs can increase. As measured by this study, levels of Tregsrecovered in the IFL and FOLFOX patietnts within 14 and 10 weeksfollowing chemotherapy, respectively.

4.3. Conclusions

-   -   Both IFL and FOLFOX treatment resulted in a reduction of Tregs.    -   Although the levels are slightly higher in the FOLFOX group of        patients the IFL group, the percentage of CD4⁺CD25⁺ Tregs in the        total CD4⁺ T cell population decreased by 30% during        chemotherapy in both TV2 trials.

4.4. Discussion

Patients with a range of different types of cancer have shown anincrease in CD4⁺CD25⁺ Tregs in peripheral blood, lymph nodes, tumourascites and tumour tissue (For review see Nomura & Sakaguci, 2005 Curr.Top. Microbiol. Immunol. 293:287). It has also been shown thatincreasing tumour burden is associated with an increase in theproportion of Tregs (Liyanage et al, 2002. J. Immunol. 169:2756-2761;Woo et al, 2001. Cancer Res. 61:4766-4772). In this study of TV2patients, CD4⁺0)25⁺ Tregs appear to be present in the peripheral bloodof most patients within the expected normal range (up to 2-10% of thetotal peripheral blood). However, this study does not address theprobability that Tregs have increased in the vicinity of the tumour(s)in these patients, locally suppressing the ability of other immune cellsto initiate or sustain a response against inappropriately expressed selfor tumour-associated antigen(s).

Both IFL and FOLFOX chemotherapies resulted in a decrease (30%) in thepercentage of CD4⁺CD25⁺ Tregs of the total CD4⁺ T cell population inperipheral blood when measured 1 week after a chemotherapy dose. Anumber of other chemotherapy treatments have been described wheredecreased levels of CD4⁺CD25⁺ Tregs in human peripheral blood wasobserved including cyclophosphamide (Ghirighelli et al, 2004; Eur. J.Immunol. 34: 336-344; Lutsiak et al, 2005. Blood 105:2862-2868), GOLF(gemcitabine [GEM], oxaliplatin, LF and FU; Correale et al, 2005. J.Clin. Oncol. 23:147-162) and temozolomide (Su et al, 2004. J. Clin.Oncol. 4:610-616). ONTAK (recombinant IL-2 diptheria toxin conjugateDAB389IL-2) has also been shown to deplete CD4⁺CD25⁺ Tregs from humanPBMCs and allow DC transfected with RNA to prime/boost immune responses(Dannull et al, 2005 J. Clin. Invest. 115: 3623-3633).

In this study of TV2 patients the decrease in CD4⁺CD25⁺ Tregs observedin peripheral blood could reflect a loss due to apoptosis/cell death, ora change in location whereby peripheral blood Tregs have been recruitedinto tissues or lymph nodes. Since these cells administer theirsuppressive affects through cell contact, they must migrate to theappropriate sites. Within secondary lymphoid tissues, Tregs must bepresent to suppress the priming of immune responses and memoryresponses. Within inflammatory sites, CD4⁺CD25⁺ Tregs must migrate topotentially hamper a variety of possible immune reactions.

More importantly, loss of function of CD4⁺CD25⁺ Tregs may be the crucialfactor rather than loss of absolute numbers of Tregs, in order to havethe capacity to initiate or sustain immune responses against self andtumour-associated antigens (Coulie and Connerotte, 2005. Curr. Opin.Immunol. 17:320-325). The mechanism by which cyclophosphamide (CY) isable to enhance immune responses by affecting Tregs has recently beenelucidated (Lutsiak et al, 2005. Blood 105:2862-2868). Not only are Tregnumbers decreased, CY inhibits their suppressive capacity by increasingapoptosis, decreasing homeostatic proliferation. CY also alters geneexpression and down-regulates GITR and FoxP3. The effects of CY on Tregsare transient whereby the absolute numbers of Tregs return topre-treatment levels 10 days after CY exposure. The transient decreaseof Tregs may be essential for successful immunotherapy, whilstmaintaining some defence against auto-immunity. It would also beinteresting to determine whether the IFL and FOLFOX affect the functionof the Tregs in similar or different ways.

Inhibitors of molecules involved in the differentiation, maturation andmaintenance of Treg cells, such as FoxP3, may be exploited in order toenhance immune responses to a tumour antigen. Inhibitors of FoxP3include anti-sense treatment of its' mRNA (Veldman et al, 2006. J.Immunol. 176:3215-3222) and factors that disrupt its transcription viathe transcription factor NFAT, such as cyclosporine A (Mantel et al,2006J. Immunol. 3593-360). Interactions of FoxP3 protein with targetgenes also involves NFAT (Wu etal., 2006. Cell 126:375-387). Dopamine asalso been shown to inhibit Treg suppressive activity (Kipnis et al 2004J. Neurosci. 24:6133-6143)

Although deletion of Treg cells enhances tumour immune responses,complete absence of Treg cells is not sufficient to treat establishedtumours expressing self antigen (Antony and Restifo, 2002. J.Immunother. 25:202; Antony et al, 2005. J. Immunol. 174:2591-2601). Thissuggests that tumour regression ideally involves a decrease in Tregscombined with the generation of effector T cells. Hence, strategies thatcombine vaccination with a factor that causes the loss of Treg and/orTreg function may be ideal for tumour immunotherapy.

The study of frozen PBMCs from TV2 patients measured only one type ofregulatory T cell, CD4⁺CD25⁺ Tregs. A decrease of 30% was observedduring chemotherapy in both trials. CD4⁺CD25^(hi+) T regs may originatefrom the thymus (natural Tregs) or from the periphery (adaptive Tregs;Bluestone and Abbas, 2003). There is no evidence to suggest that Tregsthat suppress immune responses against self antigens arise only from thethymus, or only from the periphery. Ablation of thymic-derived Tregs mayresult in a decrease of Tregs for a few weeks before they can beregenerated from the appropriate bone marrow precursors.

APPENDIX ONE Patient Demographics

TV2 FOLFOX Age (Years at trial Subject Number entry) Gender Race 101 68Male White 102 65 Male White 103 65 Male White 104 59 Female White 10556 Male White 106 54 Female White 107 67 Male White 108 59 Male White109 66 Male Black 110 55 Male White 111 65 Female White 112 72 FemaleWhite 113 47 Male Asian 114 56 Female White 115 62 Male White 116 49Female White 117 51 Male White

TV2 IFL Age (Years at trial Subject Number entry) Gender Race 001 60Female White 002 65 Male White 003 66 Male White 004 55 Male Black 00558 Male White 006 66 Male White 007 62 Female White 008 68 Female White009 64 Female White 010 49 Female White 011 66 Male White 012 64 MaleWhite 013 65 Female White 014 73 Male White 015 46 Male White 016 63Male White 017 62 Male White 018 61 Male White 019 62 Male White

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1-20. (canceled)
 21. A method of treating cancer in a subject, said method comprising: administering chemotherapy to the subject, and immunizing the subject with an antigen up to 6 weeks after the chemotherapy, wherein the antigen is 5T4 and wherein the chemotherapy is selected from the group consisting of irinotecan, fluorouracil, leucovorin, FOLFOX (5-fluorouracil, leucovorin and oxaliplatin), and IFL (irinotecan, 5-fluorouracil and leucovorin).
 22. The method of claim 21, comprising immunizing the subject with the antigen prior to administering the chemotherapy.
 23. The method of claim 21, comprising immunizing the subject with the antigen during a same time frame as administering the chemotherapy.
 24. A method for treating cancer in a subject, said method comprising: administering chemotherapy to the subject; and immunizing the subject with an antigen within 48 hours after chemotherapy, wherein the antigen is 5T4.
 25. A method for treating cancer in a subject, said method comprising: administering chemotherapy to the subject; and immunizing the subject with an antigen between 4 and 6 weeks after chemotherapy, wherein the antigen is 5T4. 